FAP-Activated Therapeutic Agents, and Uses Related Thereto

ABSTRACT

Disclosed are prodrugs of anthracyclines (such as doxorubicin) and derivatives thereof that are selectively cleaved and activated by fibroblast activating protein (FAP). The prodrugs are useful for targeted delivery of “warhead” anthracycline or anthracycline derivative to FAP-expressing tissues, including cancer (e.g., solid tumors). Also provided are pharmaceutical compositions comprising the prodrugs, as well as methods of using the prodrugs to treat a disorder characterized by FAP upregulation, e.g., cancer, undesirable fibrosis, and undesirable inflammation.

RELATED APPLICATION

This application claims benefit of U.S. Provisional Patent ApplicationNo. 62/012,003, filed Jun. 13, 2014.

BACKGROUND

Cancer is characterized by cell proliferation without normal regulationby external signals, and the potential to invade and metastasize toother tissues. For many years chemotherapy has been a mainstay oftreatment for various types of cancer. Conventional chemotherapy worksessentially by poisoning rapidly dividing cells. As such, it hasrelatively low selectivity for cancer cells per se, resulting in thefamiliar side effects of hair loss, diarrhea and other forms ofgastrointestinal upset, and marrow suppression. Such off-target sideeffects frequently become dose-limiting, and typically impose aconstraint on treatment efficacy.

For example, doxorubicin, also known as hydroxydaunorubicin, is a drugused in cancer chemotherapy. It is an anthracycline antibiotic, closelyrelated to the natural product daunomycin. Like all anthracyclines, itworks by intercalating DNA, with the most serious adverse effect beinglife-threatening heart damage. Doxorubicin is commonly used in thetreatment of a wide range of cancers, including hematologicalmalignancies, many types of carcinoma, and soft tissue sarcomas.

Anticancer therapy would be greatly improved if it were selectivelytargeted to cancer cells. Many approaches have been proposed anddeveloped with the goal of achieving selective targeting of cancertreatment agents. For example, cytotoxic agents have been linked tomonoclonal antibodies and antigen-specific fragments thereof which arecapable of binding specifically to certain tumor antigens.

The effect of folate-targeted liposomal doxorubicin (FTL-Dox) has beenwell characterized in folate receptor (FR)-overexpressing tumors invitro, particularly in KB human carcinoma cells. Riviere et al. J DrugTargeting 19(1):14-24 (2011) investigated the antitumor activity ofFTL-Dox injected intravenously into mice bearing KB tumors. Mice wereadministered a single intravenous injection of free Dox, nontargetedPEGylated liposomal Dox (PL-Dox), or FTL-Dox. FTLs and PLs accumulatedsimilarly in tumor tissue, despite the faster clearance of FTLs fromcirculation. Mice treated with FTL-Dox (20 mg/kg) displayed greaterinhibition of tumor growth, and almost a 50 percent increase in lifespan, compared to mice receiving PL-Dox (20 mg/kg). Riviere et al.concluded that while FTLs administered systemically have the potentialto enhance the delivery of anticancer drugs in vivo, their removal byFR-expressing normal tissues may have to be blocked if the benefits oftumor targeting are to be realized.

Membrane-bound proteases have recently emerged as critical mediators oftumorigenesis, angiogenesis, and metastasis. Fibroblast activationprotein alpha (FAPα, or simply FAP; EC 3.4.21.-), also known as sepraseor 170 kDa melanoma membrane-bound gelatinase, is a homodimeric integralmembrane protein belonging to the serine protease family. Scanlan et al.(1994) Proc Natl Acad Sci USA 91:5657-61; and WO 97/34927 (incorporatedby reference).

Normal adult tissues generally do not express detectable amounts of FAP.In contrast, FAP is expressed in reactive stromal fibroblasts ofepithelial cancers, granulation tissue of healing wounds, and malignantcells of bone and soft tissue sarcomas. FAP is thought to be involved inthe control of fibroblast growth or epithelial-mesenchymal interactionsduring development, tissue repair, and epithelial carcinogenesis.Significantly, most common types of epithelial cancers, including morethan 90 percent of breast, non-small cell lung, and colorectalcarcinomas, contain FAP-expressing stromal fibroblasts. Scanlan et al.Proc Natl Acad Sci USA 91:5657-61 (1994). Because in adults itsexpression is restricted to pathologic sites, including cancer,fibrosis, arthritis, wounding, and inflammation, FAP can provide targetspecificity to therapeutic agents.

U.S. Pat. No. 6,613,879 (incorporated by reference) to Firestone et al.discloses a prodrug that is capable of being converted into a cytotoxicor cytostatic drug by catalytic action of human FAP. The prodrugincludes a cleavage site which is recognized by FAP.

PCT Publication WO 2013/033396 (incorporated by reference) discloses aFAP-activated prodrug of a proteasome inhibitor, wherein the proteasomeinhibitor is linked to a FAP substrate, such that when the proteasomeinhibitor is released from the prodrug as a result of cleavage by FAP,the proteasome inhibitor inhibits the proteolytic activity of aproteasome with a Ki of 500 nM or less.

SUMMARY OF THE INVENTION

One aspect of the invention is a prodrug of an anthracycline oranthracycline derivative which is selectively cleaved and activated byfibroblast activating protein (FAP). In certain embodiments, theinvention provides prodrugs of doxorubicin which are selectively cleavedand activated by FAP. In certain embodiments, the invention providesprodrugs of anthracyclines (such as doxorubicin) which are selectivelycleaved and activated by FAP relative to (i.e., but not by) prolylendopeptidase EC 3.4.21.26 (PREP).

In certain embodiments, the invention provides a prodrug for fibroblastactivation protein (FAP)-dependent release of an active drug agent,comprising an FAP substrate covalently linked to a drug agent via a bondor a self-immolative linker. Upon cleavage by FAP of the FAP substrate,the drug agent is released in its active form or in a form that isreadily metabolized to its active form. The prodrug has less than 50% ofthe therapeutic activity of the active form of the drug agent, and morepreferably less than 60%, 70%, 80%, 90%, 95%, or even 98%. The FAPsubstrate has a k_(cat)/K_(m) for cleavage by FAP at least 10-foldgreater than for cleavage by prolyl endopeptidase (EC 3.4.21.26; PREP),and even more preferably at least 100-fold, 1000-fold, 5000-fold, oreven 10,000-fold greater k_(cat)/K_(m).

In certain embodiments, the prodrug may be further characterized by oneor more of the following features:

-   -   the prodrug has a therapeutic index that is at least 2 times        greater than the therapeutic index of the agent, and more        preferably at least 5, 10, 50, 100, 250, 500, 1000, 5000, or        even 10,000 times greater;    -   a larger percentage of the active drug agent is localized in the        target tissue, i.e., the tissue expressing FAP, relative to the        administration of the agent alone, when compared on an        equivalent dose basis—i.e., the ratio of active drug agent        localized to the target tissue relative to other tissue (such as        blood, liver or heart) is at least 2 times greater for an        equivalent dose of the prodrug relative to the agent alone, and        preferably at least 5, 10, 100, or even 1000 times greater;    -   the maximum tolerated dose of the prodrug is at least 2 times        greater than the maximum tolerated dose of the agent alone, and        even more preferably at least 5, 10, 100, or even 1000 times        greater;    -   the cell permeability of the prodrug is at least 50% less than        the cell permeability of the agent, and even more preferably at        least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 99.9% less;        and/or    -   the circulating half-life of the prodrug is at least 25% longer        than the circulating half-life of the agent alone, and even more        preferably at least 50%, 75%, 100%, 150%, 200%, 500%, 750%, or        even 1000% longer.

In one embodiment, the prodrug is represented by the general formula

or a pharmaceutically acceptable salt thereof, wherein:

R¹ represents (C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy (e.g., tert-butyloxy),(C₁-C₁₀)alkyl-C(O)—(C₁-C₁₀)alkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)cycloalkyl(C₁-C₁₀)alkyl, aryl, aryl(C₁-C₁₀)alkyl, heteroaryl, orheteroaryl(C₁-C₁₀)alkyl, wherein any R¹ is optionally substituted withone or more substituents independently selected from the groupconsisting of halo, hydroxy, carboxylate, cyano, amino, nitro, and thio(—SH); or —C(═X)R¹ represents an N-terminally blocked alpha amino acidresidue and X is O;

R² represents H or a (C₁-C₆)alkyl;

R³ represents H or a (C₁-C₆)alkyl;

R⁴ is absent or represents one, two, or three substituents, eachindependently selected from the group consisting of (C₁-C₆)alkyl, —OH,—NH₂, and halogen;

X represents O or S;

Cyt′, alone or in combination with -L-NH, represents an anthracycline orderivative thereof, less a hydrogen atom; and

L represents a 4-8 member ring or a large hydrophobic group which ispart of the anthracycline or derivative thereof and is recognized by FAPas a P′₁ residue; or L is a self-immolative linker which is metabolizedafter FAP cleavage to release Cyt′,

wherein the prodrug is selectively converted in vivo to theanthracycline or derivative thereof by FAP⁺ stromal cells.

Without meaning to be bound to any particular theory or mechanism ofaction, the inventors believe the prodrugs disclosed herein are cleavedin situ by FAP to release anthracycline or related compounds, which thenundergo spontaneous transformation into anthracycline or relatedcompounds, thereby achieving targeted delivery to FAP-expressing cellsof the anthracycline or anthracycline derivative.

An aspect of the invention is a prodrug represented by Formula I

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ represents (C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy (e.g.,        tert-butyloxy), (C₁-C₁₀)alkyl-C(O)—(C₁-C₁₀)alkyl,        (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₁₀)alkyl, aryl,        aryl(C₁-C₁₀)alkyl, heteroaryl, or heteroaryl(C₁-C₁₀)alkyl,        wherein any R¹ is optionally substituted with one or more        substituents independently selected from the group consisting of        halo, hydroxy, carboxylate, cyano, amino, nitro, and thio (—SH);        or —C(X)R¹ represents an N-terminally blocked alpha amino acid        residue, wherein X is O;    -   R² represents H or a (C₁-C₆)alkyl;    -   R³ represents H or a (C₁-C₆)alkyl;    -   R⁴ is absent or represents a (C₁-C₆)alkyl, —OH, —NH₂, or        halogen;    -   X represents O or S;    -   L represents a bond, or —N(H)-L- represents a self-immolative        linker; and    -   Cyt′ represents a radical of an anthracycline or derivative        thereof.

An aspect of the invention is a pharmaceutical composition, comprising aprodrug of the invention, or a pharmaceutically acceptable salt thereof;and a pharmaceutically acceptable carrier.

An aspect of the invention is a method of treating a disordercharacterized by fibroblast activation protein (FAP) upregulation,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a prodrug of the invention, or a pharmaceuticallyacceptable salt thereof.

In an embodiment, the disorder characterized by FAP upregulation isselected from the group consisting of cancer (e.g., solid tumors),fibrosis, and inflammation.

An aspect of the invention is a method of treating cancer, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a prodrug of the invention, or a pharmaceutically acceptablesalt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts 3099DOX and its activation by FAP.

FIG. 2 is a graph depicting in vitro enzyme kinetics of 3099DOXactivation by FAP and PREP.

FIG. 3 is a graph depicting rate of recombinant FAP activity ondoxorubicin prodrugs. GP-DOX refers to z-GP-DOX.

FIG. 4 is a bar graph depicting specificity of 3099DOX activation byFAP.

FIG. 5 is a graph depicting in vitro activation of 3099DOX and z-GP-DOXin mouse plasma.

FIG. 6 is a bar graph depicting in vitro stability of 3099DOX in normalmouse muscle lysate. Data for z-GP-DOX is shown for comparison. Datashown is following 12 h digestion at 37° C.

FIG. 7 is a graph depicting pharmacokinetics of 3099DOX (“Prodrug”) anddoxorubicin (“Warhead”) following single administration of 20 mg/kg3099DOX by iv injection in normal mouse.

FIG. 8 is a graph depicting in vivo plasma doxorubicin (DOX)concentrations following injection of 20 mg/kg 3099DOX (triangles) or 8mg/kg doxorubicin (circles); mpk, mg/kg.

FIG. 9 is a graph depicting in vivo tissue distribution of doxorubicin(DOX; “Warhead”) and 3099DOX (“Prodrug”) in mice used in the HEK-FAPtumor model study described in the examples. Samples were obtained 1hour following iv dosing with 6 mg/kg 3099DOX.

FIG. 10 is a graph depicting in vivo tumor growth in the HEK-FAP tumormodel study described in the examples. ***, p<0.05 versus vehicle; ns,not significant versus vehicle; mpk, mg/kg.

FIG. 11 is a graph depicting survival in the HEK-FAP tumor model studydescribed in the examples; mpk, mg/kg.

FIG. 12 shows the structure of doxorubicin prodrug 5057DOX, and a graphshowing that the doxorubicin prodrug is selectively activated by FAP ascompared to PREP.

FIG. 13A is a graph depicting FAP activity in mouse liver metastases.

FIG. 13B is a graph depicting FAP activity in mouse liver metastases.

FIG. 14A is a graph depicting activation kinetics for 3099DOX.

FIG. 14B is a graph depicting activation kinetics for 5057DOX.

FIG. 15 is a graph depicting pharmacodynamics of inhibition of plasmaFAP activity by 3099DOX and 5057DOX. Circles, 20 mg/kg 3099DOX; squares,80 mg/kg 3099DOX; triangles, 20 mg/kg 5057DOX; inverted triangles, 80mg/kg 5057DOX.

FIG. 16A is graph depicting plasma pharmacokinetics of prodrug in normalmice treated with the indicated doses of 5057DOX or 3099DOX; mpk, mg/kg.

FIG. 16B is graph depicting plasma pharmacokinetics of “warhead” innormal mice treated with the indicated doses of 5057DOX or 3099DOX; mpk,mg/kg.

FIG. 17 is four graphs depicting tissue distribution of 5057DOX and“warhead” in tumor-bearing HEK-FAP mice at the indicated times followingintravenous injection with 2 mg/kg of 5057DOX. Dashed lines representapproximate plasma concentrations of 5057DOX or “warhead” derived frompharmacokinetic studies in normal mice.

FIG. 18 is a graph depicting efficacy of 9 mg/kg 5057DOX vs. vehicle inHEK-FAP mice, where animals with tumors >200 mm³ on day 33 postinoculation (i.e., at start of treatment) are excluded.

FIG. 19 is a graph depicting efficacy of 9 mg/kg 5057DOX (squares) vs.vehicle and efficacy of 9 mg/kg 3099DOX (inverted triangles) vs. vehicleHEK-FAP mice, where animals with tumors >200 mm³ on day 33 postinoculation (i.e., at start of treatment) are excluded.

DETAILED DESCRIPTION OF THE INVENTION

Fibroblast activation protein (FAP) is a post-prolyl cleaving serineprotease belonging to the dipeptidyl peptidase (DPP-IV)-like subfamily.FAP and prolyl endopeptidase (PREP; EC 3.4.21.26) are the only knownmammalian proteases that can cleave on the C-terminal side of aninternal proline residue. FAP's P₄-P₁ cleavage specificity requiresproline at P₁, and glycine or a D-amino acid at P₂, prefers smalluncharged amino acids at P₃, and tolerates most amino acids at P₄. PREP,unlike FAP, is constitutively and ubiquitously expressed.

The invention exploits the enzymatic activity and specificity of FAP,and the properties of self-immolative linkers, to provide non-cytotoxic,non-cytostatic prodrugs that are capable of targeting delivery ofanthracycline compounds to FAP-expressing cells, e.g., reactive stromalfibroblasts of epithelial cancers, granulation tissue of healing wounds,and malignant cells of bone and soft tissue sarcomas.

An aspect of the invention is a prodrug represented by Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ represents (C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy (e.g., tert-butyloxy),(C₁-C₁₀)alkyl-C(O)—(C₁-C₁₀)alkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)cycloalkyl(C₁-C₁₀)alkyl, aryl, aryl(C₁-C₁₀)alkyl, heteroaryl, orheteroaryl(C₁-C₁₀)alkyl, wherein any R¹ is optionally substituted withone or more substituents independently selected from the groupconsisting of halo, hydroxy, carboxylate, cyano, amino, nitro, and thio(—SH); or —C(X)R¹ represents an N-terminally blocked alpha amino acidresidue, wherein X is O;

R² represents H or a (C₁-C₆)alkyl;

R³ represents H or a (C₁-C₆)alkyl;

R⁴ is absent or represents a (C₁-C₆)alkyl, —OH, —NH₂, or halogen;

X represents O or S;

L represents a bond, or —N(H)-L- represents a self-immolative linker(e.g., —NH—(CH₂)₄—C(O)— or —NH—(CH₂)₃—C(O)—); and

Cyt′ represents a radical of an anthracycline or derivative thereof.

Anthracyclines and analogs thereof specifically include, withoutlimitation, doxorubicin, daunorubicin, epirubicin, idarubicin,pirarubicin, valrubicin, aclarubicin, mitoxantrone, actinomycin,bleomycin, plicamycin, and mitomycin. For example, the anthracyclinemoiety can be represented by the formula

wherein,

R^(c) represents (C₁-C₆)alkyl, (C₁-C₆)hydroxyalkyl, or(C₁-C₆)alkanoyloxy(C₁-C₆)alkyl, in particular methyl, hydroxymethyl,diethoxyacetoxymethyl, or butyryloxymethyl;

R^(d) represents hydrogen, hydroxyl, or (C₁-C₆)alkoxy, in particularmethoxy;

one of R^(e) and R^(f) represents a hydrogen atom; and the otherrepresents a hydrogen atom or a hydroxy or tetrahydropyrany-2-yloxy(OTHP) group.

In certain embodiments, Cyt′ represents a radical of an anthracycline oranalog thereof. In certain embodiments, Cyt′ represents a radical ofdoxorubicin.

In certain embodiments, L represents a bond.

In an embodiment, L represents a bond, and Cyt′ represents a radical ofdoxorubicin.

In certain embodiments, —N(H)-L- represents a self-immolative linker.For example, in one embodiment, the self-immolative linker is—NH—(CH₂)₄—C(O)—. In one embodiment, the self-immolative linker is—NH—(CH₂)₃—C(O)—.

In certain embodiments, the self-immolative linker isp-aminobenzyloxycarbonyl (PABC).

In certain embodiments, the self-immolative linker is2,4-bis(hydroxymethyl)aniline.

In an embodiment, —N(H)-L- represents a self-immolative linker, and Cyt′represents a radical of doxorubicin.

In certain embodiments, R¹ represents (C₁-C₁₀)alkyl, optionallysubstituted with one or more substituents independently selected fromthe group consisting of halo, hydroxy, carboxylate, cyano, amino, nitro,and thio (—SH).

In certain embodiments, R¹ represents (C₁-C₁₀)alkoxy, optionallysubstituted with one or more substituents independently selected fromthe group consisting of halo, hydroxy, carboxylate, cyano, amino, nitro,and thio (—SH). For example, in certain embodiments, R¹ representsmethoxy. As another example, in certain embodiments, R¹ representstert-butyloxy.

In certain embodiments, R¹ represents (C₁-C₁₀)alkyl-C(O)—(C₁-C₁₀)alkyl,optionally substituted with one or more substituents independentlyselected from the group consisting of halo, hydroxy, carboxylate, cyano,amino, nitro, and thio (—SH).

In certain embodiments, R¹ represents (C₃-C₈)cycloalkyl, optionallysubstituted with one or more substituents independently selected fromthe group consisting of halo, hydroxy, carboxylate, cyano, amino, nitro,and thio (—SH). For example, in certain embodiments, R representscyclopropyl.

In certain embodiments, R¹ represents (C₃-C₈)cycloalkyl(C₁-C₁₀)alkyl,optionally substituted with one or more substituents independentlyselected from the group consisting of halo, hydroxy, carboxylate, cyano,amino, nitro, and thio (—SH).

In certain embodiments, R¹ represents aryl, optionally substituted withone or more substituents independently selected from the groupconsisting of halo, hydroxy, carboxylate, cyano, amino, nitro, and thio(—SH). For example, in certain embodiments, R¹ represents phenyl.

In certain embodiments, R¹ represents aryl(C₁-C₁₀)alkyl, optionallysubstituted with one or more substituents independently selected fromthe group consisting of halo, hydroxy, carboxylate, cyano, amino, nitro,and thio (—SH). For example, in certain embodiments, R¹ representsbenzyl.

In certain embodiments, R¹ represents heteroaryl, optionally substitutedwith one or more substituents independently selected from the groupconsisting of halo, hydroxy, carboxylate, cyano, amino, nitro, and thio(—SH). In certain embodiments, R¹ represents an N-containing heteroaryl,optionally substituted with one or more substituents independentlyselected from the group consisting of halo, hydroxy, carboxylate, cyano,amino, nitro, and thio (—SH). In certain embodiments, R¹ represents anO-containing heteroaryl, optionally substituted with one or moresubstituents independently selected from the group consisting of halo,hydroxy, carboxylate, cyano, amino, nitro, and thio (—SH). In certainembodiments, R¹ represents an S-containing heteroaryl, optionallysubstituted with one or more substituents independently selected fromthe group consisting of halo, hydroxy, carboxylate, cyano, amino, nitro,and thio (—SH).

In certain embodiments, R¹ represents heteroaryl(C₁-C₁₀)alkyl,optionally substituted with one or more substituents independentlyselected from the group consisting of halo, hydroxy, carboxylate, cyano,amino, nitro, and thio (—SH). In certain embodiments, R¹ represents anN-containing heteroaryl(C₁-C₁₀)alkyl, optionally substituted with one ormore substituents independently selected from the group consisting ofhalo, hydroxy, carboxylate, cyano, amino, nitro, and thio (—SH). Incertain embodiments, R¹ represents an O-containingheteroaryl(C₁-C₁₀)alkyl, optionally substituted with one or moresubstituents independently selected from the group consisting of halo,hydroxy, carboxylate, cyano, amino, nitro, and thio (—SH). In certainembodiments, R¹ represents an S-containing heteroaryl(C₁-C₁₀)alkyl,optionally substituted with one or more substituents independentlyselected from the group consisting of halo, hydroxy, carboxylate, cyano,amino, nitro, and thio (—SH).

In certain embodiments, R¹ represents a radical of a cyclic aromaticmoiety, preferably a mono-, bi-, or tri-cyclic including from 5-12 ringatoms, and is preferably a cyclic heteroaromatic including, for example,1-4 nitrogen atoms, and even more preferably is a basic cyclicheteroaromatic moiety including at least one lone pair of electrons thatis not part of the aromatic system (i.e., may extend in the plane of thering), such as quinoline and isoquinoline, though may also be aheteroaromatic ring containing basic as well as non-basic nitrogenatoms, e.g., imidazole or purine.

In certain embodiments, R¹ represents quinolinyl.

In certain embodiments, R¹ represents isoquinolinyl.

In certain embodiments, —C(X)R¹ represents an N-terminally blocked alphaamino acid residue, wherein X is O.

In certain embodiments, R² represents H.

In certain other embodiments, R² represents a (C₁-C₆)alkyl. For example,in certain embodiments, R² represents methyl.

In certain embodiments, R³ represents H.

In certain other embodiments, R³ represents a (C₁-C₆)alkyl. For example,in certain embodiments, R³ represents methyl, ethyl, propyl, orisopropyl. In certain embodiments, R³ is methyl.

In certain embodiments, R⁴ is absent.

In certain other embodiments, R⁴ represents a (C₁-C₆)alkyl. For example,in certain embodiments, R⁴ represents methyl.

In certain embodiments, R⁴ represents —OH.

In certain embodiments, R⁴ represents —NH₂.

In certain embodiments, R⁴ represents halogen. For example, in certainembodiments, R⁴ represents F. As another example, in certainembodiments, R⁴ represents Cl.

In certain embodiments, X represents O.

In certain other embodiments, X represents S.

In certain embodiments, L is a bond, and the anthracycline oranthracycline derivative is selected from doxorubicin, daunorubicin,epirubicin, idarubicin, pirarubicin, aclarubicin, mitoxantrone,actinomycin, bleomycin, plicamycin, or mitomycin.

In certain embodiments, the anthracycline is doxorubicin.

In certain embodiments, —C(X)R¹ is a moiety which, at physiological pH,reduces cell permeability of the prodrug relative to the anthracyclineor anthracycline derivative. For example, in various embodiments thecell permeability for the prodrug is less than: 10 percent, 20 percent,30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 75 percent,80 percent, 85 percent, 90 percent, or 95 percent of the cellpermeability for the anthracycline or anthracycline derivative. Incertain embodiments the cell permeability for the prodrug is less than50 percent of the cell permeability for the anthracycline oranthracycline derivative.

In certain embodiments, —C(X)R¹ comprises one or more functional groupsthat are ionized at physiological pH.

In certain embodiments, —C(X)R¹ is an acyl(C₁-C₁₀)alkyl substituted withone or more functional groups that are ionized at physiological pH.

In certain embodiments, —C(X)R¹ is represented by the formulaHO₂C—(C₁-C₁₀)alkyl-C(O)—.

For example, in certain embodiments, —C(X)R¹ is represented by theformula HO₂C—(CH₂)₂—C(O)—.

In certain embodiments, —C(X)R¹ is selected from the group consisting offormyl, dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl, andmethoxysuccinyl.

In certain embodiments, —C(X)R¹ is formyl.

In certain embodiments, —C(X)R¹ is dansyl.

In certain embodiments, —C(X)R¹ is acetyl.

In certain embodiments, —C(X)R¹ is benzoyl.

In certain embodiments, —C(X)R¹ is trifluoroacetyl.

In certain embodiments, —C(X)R¹ is succinyl.

In certain embodiments, —C(X)R¹ is methoxysuccinyl.

In certain embodiments, —C(X)R¹ is selected from the group consisting ofaryl(C₁-C₆)acyl and heteroaryl(C₁-C₆)acyl.

In certain embodiments, —C(X)R¹ is an aryl(C₁-C₆)acyl.

In certain embodiments, —C(X)R¹ is a heteroaryl(C₁-C₆)acyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁-C₆)acyl substitutedwith an aryl selected from the group consisting of benzyl, naphthalenyl,phenanthrenyl, phenolyl, and anilinyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁-C₆)acyl substitutedwith benzyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁-C₆)acyl substitutedwith naphthalenyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁-C₆)acyl substitutedwith phenanthrenyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁-C₆)acyl substitutedwith phenolyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁-C₆)acyl substitutedwith anilinyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁)acyl substitutedwith an aryl selected from the group consisting of benzyl, naphthalenyl,phenanthrenyl, phenolyl, and anilinyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁)acyl substitutedwith benzyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁)acyl substitutedwith naphthalenyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁)acyl substitutedwith phenanthrenyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁)acyl substitutedwith phenolyl.

In certain embodiments, the aryl(C₁-C₆)acyl is a (C₁)acyl substitutedwith anilinyl.

In certain embodiments, —C(X)R¹ is a heteroaryl(C₁-C₆)acyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with a heteroaryl selected from the group consisting ofpyrryl, furyl, thiophenyl (a/k/a thienyl), imidazolyl, oxazolyl,thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, andpyrimidinyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with pyrryl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with furyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with thiophenyl (a/k/a thienyl).

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with imidazolyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with oxazolyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with thiazolyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with triazolyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with pyrazolyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with pyridinyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with pyrizinyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with pyridazinyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with pyrimidinyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with a heteroaryl selected from the group consisting ofpyrryl, furyl, thiophenyl (a/k/a thienyl), imidazolyl, oxazolyl,thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, andpyrimidinyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with pyrryl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with furyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with thiophenyl (a/k/a thienyl).

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with imidazolyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with oxazolyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with thiazolyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with triazolyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with pyrazolyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with pyridinyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with pyrizinyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with pyridazinyl.

In certain embodiments, the heteroaryl(C₁-C₆)acyl is a (C₁)acylsubstituted with pyrimidinyl.

In an embodiment, the prodrug is represented by a formula selected fromthe group consisting of:

In an embodiment, the prodrug is represented by the formula

In an embodiment, the prodrug isN—((R)-1-((R)-2-(((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)isonicotinamide,or a pharmaceutically acceptable salt thereof.

In an embodiment, the prodrug isN—((R)-1-((R)-2-(((2S,3R,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)isonicotinamide,or a pharmaceutically acceptable salt thereof.

In an embodiment, the prodrug isN—((R)-1-((R)-2-(((2S,3S,4S,6R)-6-(((1S,3S)-3-acetyl-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3-hydroxy-2-methyltetrahydro-2H-pyran-4-yl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)isonicotinamide,or a pharmaceutically acceptable salt thereof.

In an embodiment, the prodrug isN—((R)-1-((R)-2-(((2S,3S,4S,6R)-6-(((1S,3S)-3-acetyl-3,5,12-trihydroxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3-hydroxy-2-methyltetrahydro-2H-pyran-4-yl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)isonicotinamide,or a pharmaceutically acceptable salt thereof.

An aspect of the invention is a pharmaceutical composition comprising aprodrug of the invention and a pharmaceutically acceptable carrier. Inan embodiment, the pharmaceutical composition comprises two or moreprodrugs of the invention.

An aspect of the invention is a method of making a pharmaceuticalcomposition of the invention. The method includes the step of combininga compound of the invention with a pharmaceutically acceptable carrier.In an embodiment, the method further includes the step of formulatingthe pharmaceutical composition for a particular route of administration,for example, for oral administration or for intravenous administration.

An aspect of the invention relates to a packaged pharmaceutical,comprising a prodrug described herein formulated in a pharmaceuticallyacceptable excipient, in association with instructions (written and/orpictorial) describing the recommended dosage and/or administration ofthe formulation to a patient.

An aspect of the present invention relates to a method of treating adisorder characterized by fibroblast activation protein (FAP)upregulation, comprising administering to a subject in need thereof atherapeutically effective amount of a prodrug of the invention.Disorders characterized by FAP upregulation include, without limitation,cancer (e.g., solid tumors), abnormal cell proliferation, fibrosis, andinflammation. In an embodiment, the disorder characterized by FAPupregulation is selected from the group consisting of cancer, fibrosis,and inflammation.

In an embodiment, the disorder characterized by FAP upregulation iscancer (e.g., a solid tumor).

In an embodiment, the disorder characterized by FAP upregulation is abreast carcinoma.

In an embodiment, the disorder characterized by FAP upregulation is anon-small cell lung carcinoma.

In an embodiment, the disorder characterized by FAP upregulation is acolorectal carcinoma.

In an embodiment, the disorder characterized by FAP upregulation isfibrosis.

In an embodiment, the disorder characterized by FAP upregulation isinflammation.

In certain embodiments, the method of treating a disorder characterizedby FAP upregulation further comprises administering to the subject inneed thereof a therapeutically effective amount of a chemotherapeuticagent.

In certain embodiments, the method of treating a disorder characterizedby FAP upregulation further comprises administering to the subject inneed thereof a therapeutically effective amount of an anti-inflammatoryagent.

An aspect of the present invention relates to a method of treatingcancer, comprising administering to a subject in need thereof atherapeutically effective amount of a prodrug of the invention.

In an embodiment, the cancer is a breast carcinoma.

In an embodiment, the cancer is a non-small cell lung carcinoma.

In an embodiment, the cancer is a colorectal carcinoma.

In certain embodiments, the method of treating cancer further comprisesadministering to the subject in need thereof a therapeutically effectiveamount of a chemotherapeutic agent.

DEFINITIONS

In the context of this invention, a “drug” shall mean a chemicalcompound that may be administered to humans or animals as an aid in thetreatment of disease. In particular, a drug is an active pharmaceuticalagent.

As used herein, “physiological pH” means tissue or blood pH compatiblewith life. Physiological pH is typically 6.8 to 8.4. In one embodiment,physiological pH is 7.0 to 8.0. In one embodiment, physiological pH is7.2 to 7.8.

The term “treat” or “treating” as used herein means prevent, slow orhalt the progression of, reduce at least one symptom of, and/oreliminate a disease or condition of a subject. In one embodiment,“treat” or “treating” means slow or halt the progression of, reduce atleast one symptom of, and/or eliminate a disease or condition of asubject.

The term “subject” as used herein refers to a living mammal. In anembodiment, a subject is a mouse, rat, hamster, guinea pig, rabbit, cat,dog, goat, sheep, pig, horse, cow, or non-human primate. In anotherembodiment, a subject is a human.

A “therapeutically effective amount” of a compound with respect to usein treatment, refers to an amount of the compound in a preparationwhich, when administered as part of a desired dosage regimen (to amammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The term “self-eliminating linker” or “self-immolative linker” refers toa temporary extender, spacer, or placeholder unit attaching two or moremolecules together by chemical bonds that are cleaved under definedconditions to release the two molecules. In general, a self-eliminatingor self-immolative linker may be linear or branched, and may link two ormore of the same molecules together, or may link two or more differentmolecules together. The self-eliminating or self-immolative linker maydegrade, decompose, or fragment under, for example, physiologicalconditions, acidic conditions, basic conditions, or in the presence ofspecific chemical agents. Examples of self-eliminating linkers include,but are not limited to, p-aminobenzyloxycarbonyl (PABC) and2,4-bis(hydroxymethyl)aniline.

The term “prodrug” as used herein encompasses compounds that, underphysiological conditions, are converted into therapeutically activeagents. A common method for making a prodrug is to include selectedmoieties that are hydrolyzed under physiological conditions to revealthe desired molecule. In other embodiments, the prodrug is converted byan enzymatic activity of the host animal. In an embodiment, a prodrughas less than 10 percent activity relative to the free or active drugderived or released therefrom. In an embodiment, a prodrug has less than5 percent activity relative to the free or active drug derived orreleased therefrom. In an embodiment, a prodrug has less than 1 percentactivity relative to the free or active drug derived or releasedtherefrom.

As used herein, a “prodrug of the invention” or a “compound of theinvention” refers to any prodrug of Formula I as disclosed herein.Except if otherwise expressly excluded, the term “prodrug of theinvention” or “compound of the invention” further encompassespharmaceutically acceptable salts of such prodrug of Formula I.

The term “pharmaceutically acceptable salt” refers to any relativelynon-toxic inorganic or organic acid addition salt of the prodrug(s).These salts can be prepared in situ during the final isolation andpurification of the prodrug(s), or by separately reacting a purifiedprodrug(s) in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts, and the like. See, for example, Berge et al.(1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salt” in these instancesrefers to any relatively non-toxic inorganic or organic base additionsalts of the prodrug(s). These salts can likewise be prepared in situduring the final isolation and purification of the prodrug(s), or byseparately reacting the purified prodrug(s) in its free acid form with asuitable base, such as the hydroxide, carbonate, or bicarbonate of apharmaceutically acceptable metal cation, with ammonia, or with apharmaceutically acceptable organic primary, secondary, or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts, andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, and the like (see, forexample, Berge et al., supra).

The phrase “pharmaceutically acceptable excipient” or “pharmaceuticallyacceptable carrier” as used herein means a pharmaceutically acceptablematerial, composition or vehicle, such as a liquid or solid filler,diluent, excipient, solvent or encapsulating material, involved incarrying or transporting the subject chemical from one organ or portionof the body, to another organ or portion of the body. Each carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation, not injurious to the patient, andsubstantially non-pyrogenic. Some examples of materials which can serveas pharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations. In certain embodiments, pharmaceutical compositions of thepresent invention are non-pyrogenic, i.e., do not induce significanttemperature elevations when administered to a patient.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “amino acid residue” or “amino acid” is intended to embrace allcompounds, whether natural or synthetic, which include both an aminofunctionality and an acid functionality, including amino acid analoguesand derivatives. In certain embodiments, the amino acids contemplated inthe present invention are those naturally occurring amino acids found inproteins, or the naturally occurring anabolic or catabolic products ofsuch amino acids, which contain amino and carboxyl groups.

Naturally occurring amino acids are identified throughout by theconventional three-letter and/or one-letter abbreviations, correspondingto the trivial name of the amino acid, in accordance with the followinglist. The abbreviations are accepted in the peptide art and arerecommended by the IUPAC-IUB commission in biochemical nomenclature.

Amino Acid Three-letter One-letter Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu EGlutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV Unknown or “other” Xaa X

The term “amino acid residue” further includes analogues, derivatives,and congeners of any specific amino acid referred to herein, as well asC-terminal or N-terminal protected amino acid derivatives (e.g.,modified with an N-terminal or C-terminal protecting group).

The term “peptide,” as used herein, refers to a sequence of amino acidresidues linked together by peptide bonds or by modified peptide bonds.The term “peptide” is intended to encompass peptide analogues, peptidederivatives, peptidomimetics and peptide variants. The term “peptide” isunderstood to include peptides of any length. Peptide sequences set outherein are written according to the generally accepted conventionwhereby the N-terminal amino acid is on the left, and the C-terminalamino acid is on the right.

The term “peptide analogue,” as used herein, refers to a peptidecomprising one or more non-naturally occurring amino acid. Examples ofnon-naturally occurring amino acids include, but are not limited to,D-amino acids (i.e., an amino acid of an opposite chirality to thenaturally occurring form), N-α-methyl amino acids, C-α-methyl aminoacids, β-methyl amino acids, β-alanine (β-Ala), norvaline (Nva),norleucine (Nle), 4-aminobutyric acid (γ-Abu), 2-aminoisobutyric acid(Aib), 6-aminohexanoic acid (ε-Ahx), ornithine (orn), hydroxyproline(Hyp), sarcosine, citrulline, cysteic acid, cyclohexylalanine, α-aminoisobutyric acid, t-butylglycine, t-butylalanine, 3-aminopropionic acid,2,3-diaminopropionic acid (2,3-diaP), D- or L-phenylglycine, D- orL-2-naphthylalanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (Tic), D- or L-2-thienylalanine (Thi), D- or L-3-thienylalanine, D-or L-1-, 2-, 3- or 4-pyrenylalanine, D- or L-(2-pyridinyl)-alanine, D-or L-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- orL-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine,D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- orL-p-biphenylalanine, D- or L-p-methoxybiphenylalanine, methioninesulphoxide (MSO) and homoarginine (Har). Other examples include D- orL-2-indole(alkyl)alanines and D- or L-alkylalanines, wherein alkyl issubstituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl,pentyl, isopropyl, iso-butyl, or iso-pentyl, and phosphono- or sulfated(e.g., —SO₃H) non-carboxylate amino acids.

Other examples of non-naturally occurring amino acids include3-(2-chlorophenyl)-alanine, 3-chloro-phenylalanine,4-chloro-phenylalanine, 2-fluoro-phenylalanine, 3-fluoro-phenylalanine,4-fluoro-phenylalanine, 2-bromo-phenylalanine, 3-bromo-phenylalanine,4-bromo-phenylalanine, homophenylalanine, 2-methyl-phenylalanine,3-methyl-phenylalanine, 4-methyl-phenylalanine,2,4-dimethyl-phenylalanine, 2-nitro-phenylalanine,3-nitro-phenylalanine, 4-nitro-phenylalanine, 2,4-dinitro-phenylalanine,1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid,1,2,3,4-tetrahydronorharman-3-carboxylic acid, 1-naphthylalanine,2-naphthylalanine, pentafluorophenylalanine, 2,4-dichloro-phenylalanine,3,4-dichloro-phenylalanine, 3,4-difluoro-phenylalanine,3,5-difluoro-phenylalanine, 2,4,5-trifluoro-phenylalanine,2-trifluoromethyl-phenylalanine, 3-trifluoromethyl-phenylalanine,4-trifluoromethyl-phenylalanine, 2-cyano-phenyalanine,3-cyano-phenyalanine, 4-cyano-phenyalanine, 2-iodo-phenyalanine,3-iodo-phenyalanine, 4-iodo-phenyalanine, 4-methoxyphenylalanine,2-aminomethyl-phenylalanine, 3-aminomethyl-phenylalanine,4-aminomethyl-phenylalanine, 2-carbamoyl-phenylalanine,3-carbamoyl-phenylalanine, 4-carbamoyl-phenylalanine, m-tyrosine,4-amino-phenylalanine, styrylalanine, 2-amino-5-phenyl-pentanoic acid,9-anthrylalanine, 4-tert-butyl-phenylalanine, 3,3-diphenylalanine,4,4′-diphenylalanine, benzoylphenylalanine, α-methyl-phenylalanine,α-methyl-4-fluoro-phenylalanine, 4-thiazolylalanine,3-benzothienylalanine, 2-thienylalanine, 2-(5-bromothienyl)-alanine,3-thienylalanine, 2-furylalanine, 2-pyridylalanine, 3-pyridylalanine,4-pyridylalanine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid,allylglycine, 2-amino-4-bromo-4-pentenoic acid, propargylglycine,4-aminocyclopent-2-enecarboxylic acid, 3-aminocyclopentanecarboxylicacid, 7-amino-heptanoic acid, dipropylglycine, pipecolic acid,azetidine-3-carboxylic acid, cyclopropylglycine, cyclopropylalanine,2-methoxy-phenylglycine, 2-thienylglycine, 3-thienylglycine,α-benzyl-proline, α-(2-fluoro-benzyl)-proline,α-(3-fluoro-benzyl)-proline, α-(4-fluoro-benzyl)-proline,α-(2-chloro-benzyl)-proline, α-(3-chloro-benzyl)-proline,α-(4-chloro-benzyl)-proline, α-(2-bromo-benzyl)-proline,α-(3-bromo-benzyl)-proline, α-(4-bromo-benzyl)-proline,α-phenethyl-proline, α-(2-methyl-benzyl)-proline,α-(3-methyl-benzyl)-proline, α-(4-methyl-benzyl)-proline,α-(2-nitro-benzyl)-proline, α-(3-nitro-benzyl)-proline,α-(4-nitro-benzyl)-proline, α-(1-naphthalenylmethyl)-proline,α-(2-naphthalenylmethyl)-proline, α-(2,4-dichloro-benzyl)-proline,α-(3,4-dichloro-benzyl)-proline, α-(3,4-difluoro-benzyl)-proline,α-(2-trifluoromethyl-benzyl)-proline,α-(3-trifluoromethyl-benzyl)-proline,α-(4-trifluoromethyl-benzyl)-proline, α-(2-cyano-benzyl)-proline,α-(3-cyano-benzyl)-proline, α-(4-cyano-benzyl)-proline,α-(2-iodo-benzyl)-proline, α-(3-iodo-benzyl)-proline,α-(4-iodo-benzyl)-proline, α-(3-phenyl-allyl)-proline,α-(3-phenyl-propyl)-proline, α-(4-tert-butyl-benzyl)-proline,α-benzhydryl-proline, α-(4-biphenylmethyl)-proline,α-(4-thiazolylmethyl)-proline, α-(3-benzo[b]thiophenylmethyl)-proline,α-(2-thiophenylmethyl)-proline, α-(5-bromo-2-thiophenylmethyl)-proline,α-(3-thiophenylmethyl)-proline, α-(2-furanylmethyl)-proline,α-(2-pyridinylmethyl)-proline, α-(3-pyridinylmethyl)-proline,α-(4-pyridinylmethyl)-proline, α-allyl-proline, α-propynyl-proline,γ-benzyl-proline, γ-(2-fluoro-benzyl)-proline,γ-(3-fluoro-benzyl)-proline, γ-(4-fluoro-benzyl)-proline,γ-(2-chloro-benzyl)-proline, γ-(3-chloro-benzyl)-proline,γ-(4-chloro-benzyl)-proline, γ-(2-bromo-benzyl)-proline,γ-(3-bromo-benzyl)-proline, γ-(4-bromo-benzyl)-proline,γ-(2-methyl-benzyl)-proline, γ-(3-methyl-benzyl)-proline,γ-(4-methyl-benzyl)-proline, γ-(2-nitro-benzyl)-proline,γ-(3-nitro-benzyl)-proline, γ-(4-nitro-benzyl)-proline,γ-(1-naphthalenylmethyl)-proline, γ-(2-naphthalenylmethyl)-proline,γ-(2,4-dichloro-benzyl)-proline, γ-(3,4-dichloro-benzyl)-proline,γ-(3,4-difluoro-benzyl)-proline, γ-(2-trifluoromethyl-benzyl)-proline,γ-(3-trifluoromethyl-benzyl)-proline,γ-(4-trifluoromethyl-benzyl)-proline, γ-(2-cyano-benzyl)-proline,γ-(3-cyano-benzyl)-proline, γ-(4-cyano-benzyl)-proline,γ-(2-iodo-benzyl)-proline, γ-(3-iodo-benzyl)-proline,γ-(4-iodo-benzyl)-proline, γ-(3-phenyl-allyl-benzyl)-proline,γ-(3-phenyl-propyl-benzyl)-proline, γ-(4-tert-butyl-benzyl)-proline,γ-benzhydryl-proline, γ-(4-biphenylmethyl)-proline,γ-(4-thiazolylmethyl)-proline, γ-(3-benzothioienylmethyl)-proline,γ-(2-thienylmethyl)-proline, γ-(3-thienylmethyl)-proline,γ-(2-furanylmethyl)-proline, γ-(2-pyridinylmethyl)-proline,γ-(3-pyridinylmethyl)-proline, γ-(4-pyridinylmethyl)-proline,γ-allyl-proline, γ-propynyl-proline,trans-4-phenyl-pyrrolidine-3-carboxylic acid,trans-4-(2-fluoro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-fluoro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(4-fluoro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-chloro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-chloro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(4-chloro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-bromo-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-bromo-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(4-bromo-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-methyl-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-methyl-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(4-methyl-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-nitro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-nitro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(4-nitro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(1-naphthyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-naphthyl)-pyrrolidine-3-carboxylic acid,trans-4-(2,5-dichloro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2,3-dichloro-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-trifluoromethyl-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-trifluoromethyl-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(4-trifluoromethyl-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-cyano-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-cyano-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(4-cyano-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-methoxy-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-methoxy-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(4-methoxy-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-hydroxy-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-hydroxy-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(4-hydroxy-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2,3-dimethoxy-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3,4-dimethoxy-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(3,5-dimethoxy-phenyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-pyridinyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-pyridinyl)-pyrrolidine-3-carboxylic acid,trans-4-(6-methoxy-3-pyridinyl)-pyrrolidine-3-carboxylic acid,trans-4-(4-pyridinyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-thienyl)-pyrrolidine-3-carboxylic acid,trans-4-(3-thienyl)-pyrrolidine-3-carboxylic acid,trans-4-(2-furanyl)-pyrrolidine-3-carboxylic acid,trans-4-isopropyl-pyrrolidine-3-carboxylic acid,4-phosphonomethyl-phenylalanine, benzyl-phosphothreonine,(1′-amino-2-phenyl-ethyl)oxirane, (1′-amino-2-cyclohexyl-ethyl)oxirane,(1′-amino-2-[3-bromo-phenyl]ethyl)oxirane,(1′-amino-2-[4-(benzyloxy)phenyl]ethyl)oxirane,(1′-amino-2-[3,5-difluoro-phenyl]ethyl)oxirane,(1′-amino-2-[4-carbamoyl-phenyl]ethyl)oxirane,(1′-amino-2-[benzyloxy-ethyl])oxirane,(1′-amino-2-[4-nitro-phenyl]ethyl)oxirane,(1′-amino-3-phenyl-propyl)oxirane, (1′-amino-3-phenyl-propyl)oxirane,and/or salts and/or protecting group variants thereof.

The term “peptide derivative,” as used herein, refers to a peptidecomprising additional chemical or biochemical moieties not normally apart of a naturally occurring peptide. Peptide derivatives includepeptides in which the amino-terminus and/or the carboxy-terminus and/orone or more amino acid side chain has been derivatised with a suitablechemical substituent group, as well as cyclic peptides, dual peptides,multimers of the peptides, peptides fused to other proteins or carriers,glycosylated peptides, phosphorylated peptides, peptides conjugated tolipophilic moieties (for example, caproyl, lauryl, stearoyl moieties)and peptides conjugated to an antibody or other biological ligand.Examples of chemical substituent groups that may be used to derivatise apeptide include, but are not limited to, alkyl, cycloalkyl and arylgroups; acyl groups, including alkanoyl and aroyl groups; esters;amides; halogens; hydroxyls; carbamyls, and the like. The substituentgroup may also be a blocking group such as Fmoc (fluorenylmethyl-O—CO—),carbobenzoxy (benzyl-O—CO—), monomethoxysuccinyl, naphthyl-NH—CO—,acetylamino-caproyl and adamantyl-NH—CO—. Other derivatives includeC-terminal hydroxymethyl derivatives, O-modified derivatives (forexample, C-terminal hydroxymethyl benzyl ether) and N-terminallymodified derivatives including substituted amides such as alkylamidesand hydrazides. The substituent group may be a “protecting group” asdetailed herein.

The term “peptidomimetic,” as used herein, refers to a compound that isstructurally similar to a peptide and contains chemical moieties thatmimic the function of the peptide. For example, if a peptide containstwo charged chemical moieties having functional activity, a mimeticplaces two charged chemical moieties in a spatial orientation andconstrained structure so that the charged chemical function ismaintained in three-dimensional space. The term peptidomimetic thus isintended to include isosteres. The term “isostere,” as used herein,refers to a chemical structure that can be substituted for a peptidebecause the steric conformation of the chemical structure is similar,for example, the structure fits a binding site specific for the peptide.Examples of peptidomimetics include peptides comprising one or morebackbone modifications (i.e., amide bond mimetics), which are well knownin the art. Examples of amide bond mimetics include, but are not limitedto, —CH₂NH—, —CH₂S—, —CH₂CH₂—, —CH═CH— (cis and trans), —COCH₂—,—CH(OH)CH₂—, —CH₂SO—, —CS—NH— and —NH—CO— (i.e., a reversed peptidebond) (see, for example, Spatola, Vega Data Vol. 1, Issue 3, (1983);Spatola, in Chemistry and Biochemistry of Amino Acids Peptides andProteins, Weinstein, ed., Marcel Dekker, New York, p. 267 (1983);Morley, J. S., Trends Pharm. Sci. pp. 463-468 (1980); Hudson et al.,Int. J. Pept. Prot. Res. 14:177-185 (1979); Spatola et al., Life Sci.38:1243-1249 (1986); Hann, J; Chem. Soc. Perkin Trans. 1, 307-314(1982); Almquist et al., J. Med Chem. 23:1392-1398 (1980);Jennings-White et al., Tetrahedron Lett. 23:2533 (1982); Szelke et al.,EP 45665 (1982); Holladay et al., Tetrahedron Lett. 24:4401-4404 (1983);and Hruby, Life Sci. 31:189-199 (1982)). Other examples ofpeptidomimetics include peptides substituted with one or morebenzodiazepine molecules (see, for example, James, G. L. et al. (1993)Science 260:1937-1942) and peptides comprising backbones cross-linked toform lactams or other cyclic structures.

The term “variant peptide,” as used herein, refers to a peptide in whichone or more amino acid residue has been deleted, added or substituted incomparison to the amino acid sequence to which the peptide corresponds.Typically, when a variant contains one or more amino acid substitutionsthey are “conservative” substitutions. A conservative substitutioninvolves the replacement of one amino acid residue by another residuehaving similar side chain properties. As is known in the art, the twentynaturally occurring amino acids can be grouped according to thephysicochemical properties of their side chains. Suitable groupingsinclude: alanine, valine, leucine, isoleucine, proline, methionine,phenylalanine and tryptophan (hydrophobic side chains); glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine (polar,uncharged side chains); aspartic acid and glutamic acid (acidic sidechains) and lysine, arginine and histidine (basic side chains). Anothergrouping of amino acids is phenylalanine, tryptophan, and tyrosine(aromatic side chains). A conservative substitution involves thesubstitution of an amino acid with another amino acid from the samegroup.

In certain embodiments, —C(X)R¹ represents an N-terminally blocked alphaamino acid residue, wherein X is O. An N-terminally blocked amino acidresidue is an amino acid residue modified by the presence of aprotecting group covalently linked to the amino group of said residue.

The phrase “protecting group” as used herein, means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations.

The term “amino-protecting group” or “N-terminal protecting group”refers to those groups intended to protect the α-N-terminal of an aminoacid or peptide or to otherwise protect the amino group of an amino acidor peptide against undesirable reactions during synthetic procedures.Commonly used N-protecting groups are disclosed in Greene, ProtectiveGroups In Organic Synthesis, (John Wiley & Sons, New York (1981)), whichis hereby incorporated by reference. Additionally, protecting groups canbe used as pro-drugs which are readily cleaved in vivo, for example, byenzymatic hydrolysis, to release the biologically active parent.α-N-protecting groups comprise lower alkanoyl groups such as formyl,acetyl (“Ac”), propionyl, pivaloyl, t-butylacetyl and the like; otheracyl groups include 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, -chlorobutyryl,benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like;sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-ethoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike; arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl,9-fluorenylmethyloxycarbonyl (Fmoc) and the like and silyl groups suchas trimethylsilyl and the like. Still other examples include theyl,succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl,benzyloxycarbonyl, methoxyazelaly, methoxyadipyl, methoxysuberyl, and2,4-dinitrophenyl.

The term “carboxy protecting group” or “C-terminal protecting group”refers to a carboxylic acid protecting ester or amide group employed toblock or protect the carboxylic acid functionality while the reactionsinvolving other functional sites of the compound are performed. Carboxyprotecting groups are disclosed in Greene, Protective Groups in OrganicSynthesis pp. 152-186 (1981), which is hereby incorporated by reference.Additionally, a carboxy protecting group can be used as a pro-drugwhereby the carboxy protecting group can be readily cleaved in vivo, forexample by enzymatic hydrolysis, to release the biologically activeparent. Such carboxy protecting groups are well known to those skilledin the art, having been extensively used in the protection of carboxylgroups in the penicillin and cephalosporin fields as described in U.S.Pat. Nos. 3,840,556 and 3,719,667, the disclosures of which are herebyincorporated herein by reference. Representative carboxy protectinggroups are C₁-C₈ lower alkyl (e.g., methyl, ethyl or t-butyl and thelike); arylalkyl such as phenethyl or benzyl and substituted derivativesthereof such as alkoxybenzyl or nitrobenzyl groups and the like;arylalkenyl such as phenylethenyl and the like; aryl and substitutedderivatives thereof such as 5-indanyl and the like; dialkylaminoalkylsuch as dimethylaminoethyl and the like); alkanoyloxyalkyl groups suchas acetoxymethyl, butyryloxymethyl, valeryloxymethyl,isobutyryloxymethyl, isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl,1-(pivaloyloxyl)-1-ethyl, 1-methyl-1-(propionyloxy)-1-ethyl,pivaloyloxymethyl, propionyloxymethyl and the like;cycloalkanoyloxyalkyl groups such as cyclopropylcarbonyloxymethyl,cyclobutylcarbonyloxymethyl, cyclopentylcarbonyloxymethyl,cyclohexylcarbonyloxymethyl and the like; aroyloxyalkyl such asbenzoyloxymethyl, benzoyloxyethyl and the like;arylalkylcarbonyloxyalkyl such as benzylcarbonyloxymethyl,2-benzylcarbonyloxyethyl and the like; alkoxycarbonylalkyl orcycloalkyloxycarbonylalkyl such as methoxycarbonylmethyl,cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl and the like;alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl such asmethoxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl,1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl and thelike; aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl,2-(5-indanyloxycarbonyloxy)ethyl and the like;alkoxyalkylcarbonyloxyalkyl such as2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like;arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl andthe like; arylalkenyloxycarbonyloxyalkyl such as2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl and the like;alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and thelike; alkylaminocarbonylaminoalkyl such asmethylaminocarbonylaminomethyl and the like; alkanoylaminoalkyl such asacetylaminomethyl and the like; heterocyclic carbonyloxyalkyl such as4-methylpiperazinylcarbonyloxymethyl and the like;dialkylaminocarbonylalkyl such as dimethylaminocarbonylmethyl,diethylaminocarbonylmethyl and the like; (5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like; and(5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like. Representativeamide carboxy protecting groups are aminocarbonyl and loweralkylaminocarbonyl groups. For example, aspartic acid may be protectedat the α-C-terminal by an acid labile group (e.g., t-butyl) andprotected at the β-C-terminal by a hydrogenation labile group (e.g.,benzyl) then deprotected selectively during synthesis. As mentionedabove, the protected carboxy group may also be a lower alkyl, cycloalkylor arylalkyl ester, for example, methyl ester, ethyl ester, propylester, isopropyl ester, butyl ester, sec-butyl ester, isobutyl ester,amyl ester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethylester and the like or an alkanoyloxyalkyl, cycloalkanoyloxyalkyl,aroyloxyalkyl or an arylalkylcarbonyloxyalkyl ester.

An “aliphatic chain” comprises the classes of alkyl, alkenyl and alkynyldefined below. A straight aliphatic chain is limited to unbranchedcarbon chain moieties. As used herein, the term “aliphatic group” refersto a straight chain, branched-chain, or cyclic aliphatic hydrocarbongroup and includes saturated and unsaturated aliphatic groups, such asan alkyl group, an alkenyl group, or an alkynyl group.

“Alkyl” refers to a fully saturated cyclic or acyclic, branched orunbranched carbon chain moiety having the number of carbon atomsspecified, or up to 30 carbon atoms if no specification is made. Forexample, alkyl of 1 to 8 carbon atoms refers to moieties such as methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, and thosemoieties which are positional isomers of these moieties. Alkyl of 10 to30 carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl,heneicosyl, docosyl, tricosyl and tetracosyl. In certain embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer.

“Cycloalkyl” means mono- or bicyclic or bridged saturated carbocyclicrings, each having from 3 to 12 carbon atoms. Likewise, preferredcycloalkyls have from 5-12 carbon atoms in their ring structure, andmore preferably have 6-10 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl,” asused herein, means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, and tert-butyl. Likewise, “lower alkenyl” and“lower alkynyl” have similar chain lengths. Throughout the application,preferred alkyl groups are lower alkyls. In certain embodiments, asubstituent designated herein as alkyl is a lower alkyl.

“Alkenyl” refers to any cyclic or acyclic, branched or unbranchedunsaturated carbon chain moiety having the number of carbon atomsspecified, or up to 26 carbon atoms if no limitation on the number ofcarbon atoms is specified; and having one or more double bonds in themoiety. Alkenyl of 6 to 26 carbon atoms is exemplified by hexenyl,heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodenyl, tridecenyl,tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl,nonadecenyl, eicosenyl, heneicosoenyl, docosenyl, tricosenyl, andtetracosenyl, in their various isomeric forms, where the unsaturatedbond(s) can be located anywherein the moiety and can have either the (Z)or the (E) configuration about the double bond(s).

“Alkynyl” refers to hydrocarbyl moieties of the scope of alkenyl, buthaving one or more triple bonds in the moiety.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur moiety attached thereto. In certain embodiments, the alkylthiomoiety is represented by one of —(S)-alkyl, —(S)-alkenyl, —(S)-alkynyl,and —(S)—(CH₂)_(m)—R¹, wherein m and R¹ are defined below.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined below, having an oxygen moiety attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propoxy,tert-butoxy, and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O— alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R¹, where m and R¹ are described below.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the formulae:

wherein R³, R⁵ and R⁶ each independently represent a hydrogen, an alkyl,an alkenyl, —(CH₂)_(m)—R¹, or R³ and R⁵ taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure; R¹ represents an alkenyl, aryl, cycloalkyl,a cycloalkenyl, a heterocyclyl, or a polycyclyl; and m is zero or aninteger in the range of 1 to 8. In certain embodiments, only one of R³or R⁵ can be a carbonyl, e.g., R³, R⁵, and the nitrogen together do notform an imide. In even more certain embodiments, R³ and R⁵ (andoptionally R⁶) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R¹. Thus, the term “alkylamine” as used hereinmeans an amine group, as defined above, having a substituted orunsubstituted alkyl attached thereto, i.e., at least one of R₃ and R₅ isan alkyl group. In certain embodiments, an amino group or an alkylamineis basic, meaning it has a conjugate acid with a pK_(a)≧7.00, i.e., theprotonated forms of these functional groups have pK_(a)s relative towater above about 7.00.

The term “aryl” as used herein includes 5- to 12-membered substituted orunsubstituted single-ring and polycyclic aromatic groups in which eachatom of the ring is carbon (i.e., carbocyclic aryl). Preferably, arylgroups include 5- to 12-membered rings, more preferably 6- to10-membered rings. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, and/or heterocyclyls. Carbocyclic aryl groupsinclude benzene, naphthalene, anthracene, phenanthrene, phenol, aniline,and the like.

The term “heteroaryl” as used herein includes 5- to 12-memberedsubstituted or unsubstituted single-ring and polycyclic aromatic groupsin which one or more atoms of the aromatic ring or ring system areheteroatoms. Preferably, heteroaryl groups include 5- to 12-memberedrings, more preferably 6- to 10-membered rings. The term “heteroaryl”also includes polycyclic ring systems having two or more cyclic rings inwhich two or more atoms are common to two adjoining rings wherein atleast one of the rings is heteroaromatic, e.g., the other cyclic ringscan be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls,and/or heterocyclyls. Heteroaryl groups include substituted orunsubstituted aromatic 5- to 12-membered ring structures, morepreferably 6- to 10-membered rings, whose ring structures include one tofour heteroatoms. Heteroaryl groups include, for example, pyrrole,furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,pyridine, pyrazine, pyridazine, pyrimidine, purine, quinoline,isoquinoline, carbazole, and the like.

The term “heterocyclyl” or “heterocyclic group” refer to 3- to12-membered ring structures, more preferably 5- to 12-membered rings,more preferably 6- to 10-membered rings, whose ring structures includeone to four heteroatoms. Heterocycles can also be polycycles.Heterocyclyl groups include, for example, thiophene, thianthrene, furan,pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring can be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl,carboxyl, silyl, sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, and the like.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the formula:

wherein X is a bond or represents an oxygen or a sulfur, and R⁷represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R¹ or apharmaceutically acceptable salt, R⁸ represents a hydrogen, an alkyl, analkenyl or —(CH₂)_(m)—R¹, where m and R¹ are as defined above. Where Xis an oxygen and R⁷ or R⁸ is not hydrogen, the formula represents an“ester.” Where X is an oxygen, and R⁷ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R⁷ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen, and R⁸ is a hydrogen, the formula represents a “formate.” Ingeneral, where the oxygen atom of the above formula is replaced by asulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R⁷ or R⁸ is not hydrogen, the formula represents a“thioester” group. Where X is a sulfur and R⁷ is a hydrogen, the formularepresents a “thiocarboxylic acid” group. Where X is a sulfur and R⁸ isa hydrogen, the formula represents a “thioformate” group. On the otherhand, where X is a bond, and R⁷ is not hydrogen, the above formularepresents a “ketone” group. Where X is a bond, and R⁷ is a hydrogen,the above formula represents an “aldehyde” group.

The term “thioxamide,” as used herein, refers to a moiety that can berepresented by the formula:

in which R^(t) is selected from the group consisting of the groupconsisting of hydrogen, alkyl, cycloalkyl, aralkyl, or aryl, preferablyhydrogen or alkyl. Moreover, “thioxamide-derived” compounds or“thioxamide analogues” refer to compounds in which one or more amidegroups have been replaced by one or more corresponding thioxamidegroups. Thioxamides are also referred to in the art as “thioamides.”

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds. It will be understood that “substitution” or “substitutedwith” includes the implicit proviso that such substitution is inaccordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br, or —I; the term “sulfhydryl” means —SH; theterm “hydroxyl” means —OH; the term “sulfonyl” means —SO₂—; the term“azido” means —N₃; the term “cyano” means —CN; the term “isocyanato”means —NCO; the term “thiocyanato” means —SCN; the term “isothiocyanato”means —NCS; and the term “cyanato” means —OCN.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the formula:

in which R³ and R⁵ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the formula:

in which R⁷ is as defined above.

The term “sulfonamide” is art recognized and includes a moiety that canbe represented by the formula:

in which R³ and R⁸ are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the formula:

in which R⁷ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the formula:

in which R¹² is selected from the group consisting of the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aralkyl, or aryl.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th ed., 1986-87, inside cover.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomer. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomer.

Conjoint Therapy

Compounds of the invention can be combined with other therapeuticagents. The compound of the invention and other therapeutic agent may beadministered simultaneously or sequentially. When the other therapeuticagents are administered simultaneously, they can be administered in thesame or separate formulations, but they are administered substantiallyat the same time. The other therapeutic agents are administeredsequentially with a compound of the invention when the administration ofthe other therapeutic agents is temporally separated from theadministration of the compound of the invention. The separation in timebetween the administration of these compounds may be a matter of minutesor it may be longer.

The compounds of the invention may also be administered in conjunctionwith an anti-cancer therapy. Anti-cancer therapies include cancermedicaments, radiation, and surgical procedures. As used herein, a“cancer medicament” refers to an agent which is administered to asubject for the purpose of treating a cancer. As used herein, “treatinga cancer” includes preventing the development of a cancer, reducing thesymptoms of cancer, and/or inhibiting the growth of an establishedcancer. In one embodiment, “treating a cancer” means reducing thesymptoms of cancer and/or inhibiting the growth of an establishedcancer, whether at a primary site or metastatic. Various types ofmedicaments for the treatment of cancer are described herein. For thepurpose of this specification, cancer medicaments are classified aschemotherapeutic agents, immunotherapeutic agents, cancer vaccines,hormone therapy, and biological response modifiers.

A chemotherapeutic agent can be selected from the group consisting ofmethotrexate, vincristine, adriamycin, cisplatin, carboplatin, non-sugarcontaining chloroethylnitrosoureas, 5-fluorouracil, mitomycin C,bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA,valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RASfarnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP,MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone,Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340,AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f,Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin,Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomaldoxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609(754)/RAS oncogene inhibitor, BMS-182751/oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer,Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed,Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin,Caelyx/liposomal doxorubicin, Fludara/Fludarabine,Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphthalimide,LU 103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine,ZD 0473/Anormed, YM 116, iodine seeds, CDK4 and CDK2 inhibitors, PARPinhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog,nitrosoureas, alkylating agents such as melphelan and cyclophosphamide,Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil,Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustine phosphatesodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU),Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, InterferonAlfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue),Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard),Mercaptopurine, Mesna, Mitotane (o.p′-DDD), Mitoxantrone HCl,Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifencitrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA),Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2,Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG),Pentostatin (2′deoxycoformycin), Semustine (methyl-CCNU), Teniposide(VM-26) and Vindesine sulfate, but it is not so limited.

An immunotherapeutic agent may be selected from the group consisting ofRibutaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225,Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210,MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447,MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT,Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5,ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab,SMART ABL 364 Ab and ImmuRAIT-CEA, but it is not so limited.

A cancer vaccine may be selected from the group consisting of EGF,anti-idiotypic cancer vaccines, gp75 antigen, GMK melanoma vaccine, MGVganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax,STn-KHL theratope, BLP25 (MUC-1), liposomal idiotypic vaccine, Melacine,peptide antigen vaccines, toxin/antigen vaccines, MVA-based vaccine,PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus and ImmuCyst/TheraCys,but it is not so limited.

The compounds of the invention may also be administered in conjunctionwith an anti-inflammatory agent. Anti-inflammatory agents includenonsteroidal anti-inflammatory drugs (NSAIDs), elemental gold,adrenocorticosteroids, vitamin D, vitamin E, and statins (HMG-Co-Areductase inhibitors). NSAIDs include, without limitation, aspirin,choline salicylate, celecoxib, diclofenac potassium, diclofenac sodium,diflunisal, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen,indomethacin, ketoprofen, magnesium salicylate, meclofenamate sodium,mefenamic acid, meloxicam, nabumetone, naproxen, naproxen sodium,oxaprozin, piroxicam, rofecoxib, salsalate, sodium salicylate, sulindac,tolmetin sodium, and valdecoxib. Adrenocorticosteroids include, withoutlimitation, betamethasone, cortisol (hydrocortisone), cortisone,dexamethasone, fludrocortisone, fluticasone, methylprednisolone,paramethasone, prednisolone, prednisone, tetrahydrocortisol, andtriamcinolone. Statins include, without limitation, atorvastatin,cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin,rosuvastatin, and simvastatin.

Dosages & Dosing Regimens

As stated above, an “effective amount” refers to any amount that issufficient to achieve a desired biological effect. Combined with theteachings provided herein, by choosing among the various activecompounds and weighing factors such as potency, relativebioavailability, patient body weight, severity of adverse side-effectsand preferred mode of administration, an effective prophylactic ortherapeutic treatment regimen can be planned which does not causesubstantial unwanted toxicity and yet is effective to treat theparticular subject. The effective amount for any particular applicationcan vary depending on such factors as the disease or condition beingtreated, the particular compound of the invention being administered,the size of the subject, or the severity of the disease or condition.One of ordinary skill in the art can empirically determine the effectiveamount of a particular compound of the invention and/or othertherapeutic agent without necessitating undue experimentation. It ispreferred generally that a maximum dose be used, that is, the highestsafe dose according to some medical judgment. Multiple doses per day maybe contemplated to achieve appropriate systemic levels of compounds.Appropriate systemic levels can be determined by, for example,measurement of the patient's peak or sustained plasma level of the drug.“Dose” and “dosage” are used interchangeably herein.

For any compound described herein the therapeutically effective amountcan be initially determined from animal models. A therapeuticallyeffective dose can also be determined from human data for compounds ofthe invention which have been tested in humans and for compounds whichare known to exhibit similar pharmacological activities, such as otherrelated active agents. Higher doses may be required for oraladministration than for parenteral administration. The applied dose canbe adjusted based on the relative bioavailability and potency of theadministered compound. Adjusting the dose to achieve maximal efficacybased on the methods described above and other methods as are well-knownin the art is well within the capabilities of the ordinarily skilledartisan.

Generally, daily intravenous doses of active compound or compounds willbe from about 0.001 milligrams/kg per day to 100 milligrams/kg per day.It is expected that intravenous doses in the range of 0.05 to 5milligrams/kg, in one or several administrations per day, will yield thedesired results. Intravenous dosing on other schedules is alsocontemplated by the invention, e.g., every-other day, semi-weekly,weekly, biweekly, and monthly. Similar dosing for other parenteralroutes of administration is also contemplated by the invention.

Generally, daily oral doses of active compound or compounds will be fromabout 0.01 milligrams/kg per day to 1000 milligrams/kg per day. It isexpected that oral doses in the range of 0.5 to 50 milligrams/kg, in oneor several administrations per day, will yield the desired results. Oraldosing on other schedules is also contemplated by the invention, e.g.,every-other day, semi-weekly, weekly, biweekly, and monthly.

Dosage may be adjusted appropriately to achieve desired drug levels,local or systemic, depending upon the mode of administration. Forexample, it is expected that intravenous administration would be from anorder to several orders of magnitude lower dose per day. In the eventthat the response in a subject is insufficient at such doses, evenhigher doses (or effective higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

Pharmaceutical Formulations & Modes of Administration

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

For use in therapy, an effective amount of the compound of the inventioncan be administered to a subject by any mode that delivers the compoundof the invention to the desired surface. Administering thepharmaceutical composition of the present invention may be accomplishedby any means known to the skilled artisan. Routes of administrationinclude but are not limited to oral, intravenous, intramuscular,intraperitoneal, subcutaneous, direct injection (for example, into atumor), mucosal, inhalation, and topical.

For oral administration, the compounds (i.e., compounds of theinvention, and other therapeutic agents) can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers, e.g., EDTA forneutralizing internal acid conditions or may be administered without anycarriers.

Also specifically contemplated are oral dosage forms of the abovecomponent or components. The component or components may be chemicallymodified so that oral delivery of the derivative is efficacious.Generally, the chemical modification contemplated is the attachment ofat least one moiety to the component molecule itself, where said moietypermits (a) inhibition of acid hydrolysis; and (b) uptake into the bloodstream from the stomach or intestine. Also desired is the increase inoverall stability of the component or components and increase incirculation time in the body. Examples of such moieties include:polyethylene glycol, copolymers of ethylene glycol and propylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. Abuchowski and Davis, “SolublePolymer-Enzyme Adducts”, In: Enzymes as Drugs, Hocenberg and Roberts,eds., Wiley-Interscience, New York, N.Y., pp. 367-383 (1981); Newmark etal., J Appl Biochem 4:185-9 (1982). Other polymers that could be usedare poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

For the component (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunum, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the compound of the invention (orderivative) or by release of the biologically active material beyond thestomach environment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic (e.g., powder); for liquid forms, a soft gelatin shell maybe used. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, thecompound of the invention (or derivative) may be formulated (such as byliposome or microsphere encapsulation) and then further contained withinan edible product, such as a refrigerated beverage containing colorantsand flavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents whichcan be used can include benzalkonium chloride and benzethonium chloride.Potential non-ionic detergents that could be included in the formulationas surfactants include lauromacrogol 400, polyoxyl 40 stearate,polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerolmonostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester,methyl cellulose and carboxymethyl cellulose. These surfactants could bepresent in the formulation of the compound of the invention orderivative either alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of the compounds of theinvention (or derivatives thereof). The compound of the invention (orderivative) is delivered to the lungs of a mammal while inhaling andtraverses across the lung epithelial lining to the blood stream. Otherreports of inhaled molecules include Adjei et al., Pharm Res 7:565-569(1990); Adjei et al., Int J Pharmaceutics 63:135-144 (1990) (leuprolideacetate); Braquet et al., J Cardiovasc Pharmacol 13(suppl. 5):143-146(1989) (endothelin-1); Hubbard et al., Annal Int Med 3:206-212 (1989)(al-antitrypsin); Smith et al., 1989, J Clin Invest 84:1145-1146(a-1-proteinase); Oswein et al., 1990, “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colo., March, (recombinant human growth hormone); Debs et al., 1988, JImmunol 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha)and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor). A method and composition for pulmonary delivery ofdrugs for systemic effect is described in U.S. Pat. No. 5,451,569,issued Sep. 19, 1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of compound of the invention (or derivative). Typically, eachformulation is specific to the type of device employed and may involvethe use of an appropriate propellant material, in addition to the usualdiluents, adjuvants and/or carriers useful in therapy. Also, the use ofliposomes, microcapsules or microspheres, inclusion complexes, or othertypes of carriers is contemplated. Chemically modified compound of theinvention may also be prepared in different formulations depending onthe type of chemical modification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise compound of the invention (orderivative) dissolved in water at a concentration of about 0.1 to 25 mgof biologically active compound of the invention per mL of solution. Theformulation may also include a buffer and a simple sugar (e.g., forcompound of the invention stabilization and regulation of osmoticpressure). The nebulizer formulation may also contain a surfactant, toreduce or prevent surface induced aggregation of the compound of theinvention caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the compound of theinvention (or derivative) suspended in a propellant with the aid of asurfactant. The propellant may be any conventional material employed forthis purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing compound of the invention (orderivative) and may also include a bulking agent, such as lactose,sorbitol, sucrose, or mannitol in amounts which facilitate dispersal ofthe powder from the device, e.g., 50 to 90% by weight of theformulation. The compound of the invention (or derivative) shouldadvantageously be prepared in particulate form with an average particlesize of less than 10 micrometers (m), most preferably 0.5 to 5 μm, formost effective delivery to the deep lung.

Nasal delivery of a pharmaceutical composition of the present inventionis also contemplated. Nasal delivery allows the passage of apharmaceutical composition of the present invention to the blood streamdirectly after administering the therapeutic product to the nose,without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

For nasal administration, a useful device is a small, hard bottle towhich a metered dose sprayer is attached. In one embodiment, the metereddose is delivered by drawing the pharmaceutical composition of thepresent invention solution into a chamber of defined volume, whichchamber has an aperture dimensioned to aerosolize and aerosolformulation by forming a spray when a liquid in the chamber iscompressed. The chamber is compressed to administer the pharmaceuticalcomposition of the present invention. In a specific embodiment, thechamber is a piston arrangement. Such devices are commerciallyavailable.

Alternatively, a plastic squeeze bottle with an aperture or openingdimensioned to aerosolize an aerosol formulation by forming a spray whensqueezed is used. The opening is usually found in the top of the bottle,and the top is generally tapered to partially fit in the nasal passagesfor efficient administration of the aerosol formulation. Preferably, thenasal inhaler will provide a metered amount of the aerosol formulation,for administration of a measured dose of the drug.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethylcellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, the compounds may alsobe formulated as a depot preparation. Such long acting formulations maybe formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer R, Science 249:1527-33(1990), which is incorporated herein by reference.

The compounds of the invention and optionally other therapeutics may beadministered per se (neat) or in the form of a pharmaceuticallyacceptable salt. When used in medicine the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof. Such salts include, but are not limited to, those prepared fromthe following acids: hydrochloric, hydrobromic, sulphuric, nitric,phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric,citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

Pharmaceutical compositions of the invention contain an effective amountof a compound of the invention and optionally therapeutic agentsincluded in a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” means one or more compatible solidor liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the compounds of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

The therapeutic agent(s), including specifically but not limited to thecompound of the invention, may be provided in particles. Particles asused herein means nanoparticles or microparticles (or in some instanceslarger particles) which can consist in whole or in part of the compoundof the invention or the other therapeutic agent(s) as described herein.The particles may contain the therapeutic agent(s) in a core surroundedby a coating, including, but not limited to, an enteric coating. Thetherapeutic agent(s) also may be dispersed throughout the particles. Thetherapeutic agent(s) also may be adsorbed into the particles. Theparticles may be of any order release kinetics, including zero-orderrelease, first-order release, second-order release, delayed release,sustained release, immediate release, and any combination thereof, etc.The particle may include, in addition to the therapeutic agent(s), anyof those materials routinely used in the art of pharmacy and medicine,including, but not limited to, erodible, nonerodible, biodegradable, ornonbiodegradable material or combinations thereof. The particles may bemicrocapsules which contain the compound of the invention in a solutionor in a semi-solid state. The particles may be of virtually any shape.

Both non-biodegradable and biodegradable polymeric materials can be usedin the manufacture of particles for delivering the therapeutic agent(s).Such polymers may be natural or synthetic polymers. The polymer isselected based on the period of time over which release is desired.Bioadhesive polymers of particular interest include bioerodiblehydrogels described in Sawhney H S et al. (1993) Macromolecules26:581-7, the teachings of which are incorporated herein. These includepolyhyaluronic acids, casein, gelatin, glutin, polyanhydrides,polyacrylic acid, alginate, chitosan, poly(methyl methacrylates),poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), andpoly(octadecyl acrylate).

The therapeutic agent(s) may be contained in controlled release systems.The term “controlled release” is intended to refer to anydrug-containing formulation in which the manner and profile of drugrelease from the formulation are controlled. This refers to immediate aswell as non-immediate release formulations, with non-immediate releaseformulations including but not limited to sustained release and delayedrelease formulations. The term “sustained release” (also referred to as“extended release”) is used in its conventional sense to refer to a drugformulation that provides for gradual release of a drug over an extendedperiod of time, and that preferably, although not necessarily, resultsin substantially constant blood levels of a drug over an extended timeperiod. The term “delayed release” is used in its conventional sense torefer to a drug formulation in which there is a time delay betweenadministration of the formulation and the release of the drug therefrom. “Delayed release” may or may not involve gradual release of drugover an extended period of time, and thus may or may not be “sustainedrelease.”

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. “Long-term” release, asused herein, means that the implant is constructed and arranged todeliver therapeutic levels of the active ingredient for at least 7 days,and preferably 30-60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES Example 1 Synthesis of 3099DOX

Synthesis of Compound 1

EDCI.HCl (2.9 g, 15 mmol), HOBt (1.6 g, 12 mmol) and DIEA (2.0 mL, 11.5mmol) were added to a solution of N-Boc-D-Ala-OH (1.9 g, 10 mmol) inanhydrous DMF (40 mL) under ice-water bath cooling. The resultingmixture was stirred at room temperature for 20 min, cooled down againwith ice-water bath and L-proline benzyl ester hydrochloride (2.54 g,10.5 mmol) was added followed by another 2.0 mL of DIEA. The reactionmixture was stirred at room temperature overnight and then condensed invacuo. The residue was dissolved with ethyl acetate (150 mL), washedsequentially by 0.1 N KHSO₄ (3×40 mL), aq. NaHCO₃ (3×40 mL), brine (30mL). The organic phase was dried over anhydrous MgSO₄, filtered, andevaporated in vacuo to give N-Boc-D-Ala-L-Pro-OBzl which was then addedto a solution of 4 N HCl in dioxane (30 mL) under ice-water cooling. Theresulting mixture was stirred at room temperature for 2 hrs and thencondensed in vacuo. The residue was co-evaporated with dichloromethane(3×30 mL) in vacuo to completely dry. Compound 1 was thus obtained as awhite powder (2.9 g, 92% over two steps).

Synthesis of Compound 2

A solution of compound 1 (2.8 g, 8.9 mmol) in anhydrous dichloromethane(100 mL) was stirred under ice-water bath cooling. DIEA (4.6 mL, 27mmol) was then added slowly followed by the addition of isonicotinoylchloride hydrochloride (1.75 g, 9.8 mmol) portion-wise over 10 min. Theresulting mixture was stirred at room temperature for 5 hrs until thereaction was complete, then diluted with more dichloromethane (100 mL),washed by water (20 mL), aq. NaHCO₃ (2×20 mL), aq. NaCl (20 mL), driedover anhydrous MgSO₄, filtered, evaporated in vacuo to give crudecompound 2 which was further purified with silica gel flash columnchromatography (CH₂Cl₂:MeOH, 20:1) to afford the pure product 2 (2.9 g,86%).

Synthesis of Compound 3

Compound 2 (1.5 g, 3.9 mmol) was added to a suspension solution of 10%Pd—C (0.15 g) in methanol (20 mL). The mixture was degassed underreduced pressure and placed under H₂ (50 psi). The mixture was stirredat room temperature for 3 hrs until the reaction was complete. Thecatalyst was then removed by filtration through Celite. The filtrate wasconcentrated in vacuo to complete dryness to give compound 3 as a whitepowder (1.05 g, 93%).

Synthesis of 3099DOX

EDCI.HCl (785 mg, 3.15 mmol) and N-hydroxysuccinimide (0.38 g, 3.3 mmol)were added to a solution of compound 3 (0.87 g, 3.0 mmol) in anhydrousDMF (25 mL) under ice-water bath cooling. The reaction mixture wasstirred at room temperature for 3 hrs, concentrated in vacuo,re-dissolved into DMSO (10 mL) to make Solution A.

A pH 7.8 phosphate buffer (50 mL) was added slowly to another solutionof Doxorubicin HCl (1.9 g, 3.3 mmol) in DMSO (140 mL) with good stirringunder ice-water bath cooling. Solution A was then added and theresulting mixture was stirred at room temperature for 8 hrs, cooled downagain with ice-water bath, diluted with water (80 mL) anddichloromethane (800 mL). The organic phase was partitioned andseparated, washed by aq. NaCl (2×200 mL), dried over anhydrous Na₂SO₄,concentrated and purified with silica gel flash column chromatography(CH₂C12:MeOH, 20:1 to 7:1) to give the pure compound 3099DOX as a littlesticky dark-red powder which was re-dissolved with 2:3 ofacetontrile-water and lyophilized to produce a good light red powder(2.0 g, 81% over two steps).

Analysis

(i) LCMS of compound 3099DOX.

-   -   0-3 min: 2% B; 3-20 min: 2-98% B; 20-25 min: 98% B.    -   Ran with 1.8 μm particle size column on the Old LCMS.    -   Calc. MW, 816; the peak at 13.5 min was observed, 839.2:[M+Na],        403.2.

(ii)¹H NMR (D₂O/ACN-d₃, 1:1) showed a 2:8 of two rotamers.

(iii)¹H NMR (CDCl₃) showed a single rotamer.

(iv) Purity Analysis

-   -   HPLC Conditions:    -   Column: Agilent Eclipse Plus C18, 4.6×50 mm, 1.8 μm particle        size    -   Column Temp: 27+2° C.    -   Sample Temp: ambient    -   Flow Rate: 0.5 mL/min    -   UV Detection Wavelength: 254 nm    -   Mobile Phase: A: 0.1% CF₃COOH in water        -   B: 0.08% CF₃COOH in acetonitrile    -   Gradient Pump Program:

Step Time Elapsed Time % A % B (minutes) (minutes) (Aqueous) (Organic) 00 98 2 3 3 98 2 17 20 2 98 5 25 2 98

-   -   The column equilibrated with the initial composition mobile        phase prior to commencing the analysis.    -   Retention time: 13.48 min; Purity: 99.39% (TAN).

(v) Stability Tests of 3099DOX (solid powder, open vials)

Remaining Purity % Test Conditions Day 0 1 week 1 month 2 month 3 month22° C./72% RH 99.4 99.3 99.5 98.1 97.5 40° C./75% RH 99.4 99.3 98.8 96.395.3

Example 2 Doxorubicin Prodrugs

Compound Structures Bioassay 3099DOX

CellTiter-Glo cell viability assay with IM-9 cells EC50 (prodrug alone)= 190 μM EC50 (prodrug + 50 nM FAP) = 430 nM EC50 (prodrug + 50 nM PREP)= 110 μM z-GP-DOX

3996DOX

CellTiter-Blue cell viability assay with CT26 cells EC50 (prodrug alone)= 190 μM EC50 (prodrug + 50 nM FAP) = 26 μM EC50 (prodrug + 50 nM PREP)= 210 μM

Example 3 Enzyme Kinetics of 3099DOX Activation by FAP

Michaelis-Menten enzyme kinetics for FAP were measured using aSpectraMax M2e microplate reader (Molecular Devices, Sunnyvale, Calif.,USA). The assays were performed in FAP buffer (50 mM Tris, 140 mM NaCl,pH 7.5) at 25° C., and the fluorescence continuously monitored atexcitation and emission wavelengths of 380 and 460 nm, respectively.Kinetic constants (k_(cat) and K_(m)) were determined using GP-AMC(Bachem, Torrance, Calif., USA), Ac-(D)-AP-AFC (MP Biomedicals, Solon,Ohio, USA) and test article concentrations equivalent to 0.1-5 timestheir respective K_(m) values, and with 5-nM enzyme. All assays wereperformed in triplicate, and the results were calculated with anonlinear regression analysis, relying on a Michaelis-Menten curve fitusing GraphPad software.

3099DOX at various concentrations ranging from 8.5×10⁻⁶ M to 6.9×10⁻⁴ Mwas incubated with 1.34×10⁻⁸ M FAP or PREP at 37° C., and releaseddoxorubicin measured. Results of kinetic analysis are shown in FIG. 2.While 3099DOX had essentially no activation with PREP, 3099DOX wasactivated by FAP with V_(max)=5.877×10⁻⁹ M/sec, K_(m)=1.12×10⁻⁵ M,k_(cat) (V_(max)/[E])=0.44 sec⁻¹, and k_(cat)/K_(m)=39171 M⁻¹ sec¹.

Example 4 Rate of Recombinant FAP Activity on Doxorubicin Prodrugs

3099DOX, z-GP-DOX, and 3996DOX were incubated in the presence ofrecombinant FAP at 37° C. for 24 hours. Released doxorubicin wasmeasured at 1, 4, 12, and 24 hours. Results are shown in FIG. 3. 3099DOXyielded free doxorubicin significantly faster than either z-GP-DOX or3996DOX, reaching maximum in approximately 8 h.

Example 5 Specificity of 3099DOX Activation by FAP

Doxorubicin prodrugs 3099DOX, z-GP-DOX, and 3996DOX, each at 100 μM,were digested 5 mg/mL FAP, PREP, or dipeptidyl peptidase IV (DPP IV) for24 hours at 37° C. Total doxorubicin was measured for each assay.Results are shown in FIG. 4. Doxorubicin was released from 3099DOX byFAP but neither PREP nor DPP IV. A similar amount of doxorubicin wasreleased from z-GP-DOX by FAP and PREP, but essentially none by DPP IV.Only about 15 percent as much doxorubicin was released from 3996DOX byFAP, but essentially none by PREP or DPP IV.

Example 6 Activation of 3099DOX in Mouse Plasma

Doxorubicin, 3099DOX, or z-PG-DOX was added to mouse plasma to producesamples containing equimolar final concentrations of 100 μM of drug orprodrug. The resulting samples were then incubated at 37° C. for 12 h,and doxorubicin was measured at 0, 4, and 12 h. Results are shown inFIG. 5. 3099DOX was activated in mouse plasma about 25 percent fasterthan was z-PG-DOX.

Example 7 Stability of 3099DOX in Mouse Muscle Lysate

3099DOX or z-PG-DOX was added to freshly prepared mouse muscle lysate toproduce samples containing equimolar final concentrations of 100 M ofprodrug. The resulting samples were then incubated at 37° C. for 12 h,and doxorubicin was measured at 12 h. Results are shown in FIG. 6.3099DOX incubated with muscle lysate released essentially nodoxorubicin, while z-GP-DOX incubated under the same conditions releaseda large amount of doxorubicin.

Example 8 Pharmacokinetics of 3099DOX in Normal Mouse

Normal, healthy mice were administered 20 mg/kg body weight 3099DOX in asingle intravenous (iv) injection. Blood was then collected at 5, 15,30, 60, 120, and 240 minutes following administration, and plasma wasprepared from each blood sample.

Plasma concentrations of prodrug (3099DOX) and doxorubicin (“warhead”)were measured by LC-MS analysis after protein crash. Results are shownin FIG. 7.

Example 9 Pharmacokinetics of Doxorubicin in Normal Mouse

Normal, healthy mice were administered either 20 mg/kg body weight3099DOX or 8 mg/kg body weight doxorubicin in a single intravenous (iv)injection. Blood was then collected at 5, 15, 30, 60, 120, and 240minutes following administration, and plasma was prepared from eachblood sample. Plasma concentrations of doxorubicin were measured byLC-MS analysis after protein crash. Results are shown in FIG. 8.

Example 10 HEK-FAP Tumor Model

Human embryonic kidney (HEK) cells were stably transfected with a mouseFAP or mock vector (Fox Chase Cancer Center, Philadelphia, Pa., USA) andcultured in RPMI 1640 cell culture medium without phenol red,supplemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate,4.5 g/L glucose, 100 I.U. penicillin, 100 μg/mL streptomycin and 1%human AB serum (VWR, Radnor, Pa., USA).

Mice were then divided into five treatment groups. Treatment startedwhen tumors were 100 mm³ in size. Animals in Group 1 received vehiclealone once per week as a single iv injection. Animals in Group 2received 2 mg/kg (3.7 mole/kg) doxorubicin once per week as a single ivinjection. Animals in Groups 3-5 received 6, 9, or 12 mg/kg (7.4, 11.1,or 14.8 mole/kg) 3099DOX, respectively, once per week as a single ivinjection.

Tumor size (volume), body weight, and survival were monitored for up to70 days after starting treatment. Additionally, tissue distribution of3099DOX and doxorubicin was measured at the end of this study.

Example 11 Tissue Distribution of 3099DOX in the HEK-FAP Tumor Model

Tissues were collected from animals in Group 3 of the HEK-FAP tumormodel study described in Example 10 1 hour after the final dose of3099DOX. Concentrations of 3099DOX (prodrug) and doxorubicin (“warhead”)were determined in heart, tumor, and plasma. Results are shown in FIG.9. Doxorubicin was concentrated in tumor tissue (ca. 300 nM) compared toheart and plasma. 3099DOX was concentrated in plasma (ca. 250 nM)compared to heart and tumor.

Example 12 Efficacy of 3099DOX in the HEK-FAP Tumor Model

Tumor growth was monitored in the HEK-FAP tumor model described inExample 10. Results are shown in FIG. 10. The mean number of days toreach 500 mm³ tumor size was 41 for vehicle-treated controls. The meannumber of days to reach 500 mm³ tumor size was 41 43 fordoxorubicin-treated Group 2. In striking contrast to either of theformer groups, the mean number of days to reach 500 mm³ tumor size was51, 57, and 63 for 3099DOX-treated Groups 3, 4, and 5, respectively. Thelatter three values were statistically significant (p<0.05) versusvehicle (Dunnett's multiple comparison test).

Example 13 Efficacy of 3099DOX in the HEK-FAP Tumor Model

Survival was also monitored in the HEK-FAP tumor model described inExample 10. Results are shown in FIG. 11. Animals treated with 3099DOXhad significantly longer survival than animals treated with eithervehicle alone or doxorubicin. As evident from FIG. 11, survival in the3099DOX-treated animals was prolonged in a dose-dependent manner.

Example 14 Cytotoxicity of FAP-Activated Doxorubicin Prodrugs AgainstVarious Tumor Lines

Cells from various tumor-derived cell lines were incubated withdoxorubicin or 3099DOX, the latter in the presence or absence of FAP,and the EC₅₀ for each was determined. Results are shown in the tablebelow.

EC₅₀ (μM) 3099DOX + 3099DOX + Cell Line DOX 3099DOX 100 μM 3099 25 nMrFAP HEK-Mock 0.05 — 280  0.04 HEK-mFAP 0.2 1.1 120 — BxPC-3 3.1 210 —2.4 HPAF-II 2.0 330 — 1.6 HT-29 0.4 340 — 0.8

Example 15 Additional FAP-Activated Doxorubicin Prodrugs

Compound Structure Bioassay 5057DOX

1. CellTiter-Blue cell viability assay with HEK- Mock cells: EC50(prodrug alone) = 480 nM EC50 (prodrug + 25 nM FAP) = 36 nM EC50(prodrug + 25 nM PREP) = 250 nM EC50 (doxorubicin alone) = 36 nM. Note:This assay involved a 72 hour, rather than the typical 48 hour,incubation of prodrug with the cells prior to addition of theCellTiter-Blue reagent. 2. CellTiter-Blue cell viability assay with HEK-Mock and HEK-mFAP cells: HEK-Mock EC50 (prodrug alone) = 460 nM EC50(prodrug + 25 nM FAP) = 26 nM EC50 (prodrug + 25 nM PREP) = 220 nM EC50(prodrug + 100 uM 2054-9) = 9.8 uM EC50 (doxorubicin alone) = 34 nMHEK-mFAP EC50 (prodrug alone) = 140 nM EC50 (prodrug + 100 uM 3099-15)= >10 uM EC50 (prodrug + 25 nM PREP) = 110 nM EC50 (prodrug + 100 uM2054-9) = 740 nM EC50 (doxorubicin alone) = 59 nM. Note: This assayinvolved a 72 hour, rather than the typical 48 hour, incubation ofprodrug with the cells prior to addition of the CellTiter-Blue reagent.3. CellTiter-Blue cell viability assay with HEK- Mock and HEK-mFAPcells: HEK-Mock EC50 (prodrug alone) = 2.3 uM EC50 (prodrug + 100 uM2054-9) = >10 uM EC50 (prodrug + 100 uM 3099-15) = >100 uM EC50(doxorubicin alone) = 95 nM HEK-mFAP EC50 (prodrug alone) = 600 nM EC50(prodrug + 100 uM 2054-9) = 5.3 uM EC50 (prodrug + 100 uM 3099-15)= >100 uM EC50 (doxorubicin alone) = 240 nM. Note: This assay involved a72 hour, rather than the typical 48 hour, incubation of prodrug with thecells prior to addition of the CellTiter-Blue reagent. 4. CellTiter-Bluecell viability assay with HEK- Mock and HEK-mFAP cells: HEK-Mock EC50(prodrug alone) = 1.1 uM EC50 (prodrug + 100 uM 5057) = >100 uM EC50(doxorubicin alone) = 92 nM HEK-mFAP EC50 (prodrug alone) = 770 nM EC50(prodrug + 100 uM 5057) = >100 uM EC50 (doxorubicin alone) = 420 nM.Note: This assay involved a 72 hour, rather than the typical 48 hour,incubation of prodrug with the cells prior to addition of theCellTiter-Blue reagent. 5. CellTiter-Blue cell viability assays: MCF-7EC50 (prodrug alone) = >100 uM EC50 (prodrug + 25 nM FAP) = 23 uM EC50(prodrug + 100 uM 5057) = >100 uM EC50 (doxorubicin alone) = 54 uMOVCAR-3 EC50 (prodrug alone) = >100 uM EC50 (prodrug + 25 nM FAP) = 1 uMEC50 (prodrug + 100 uM 5057) = >100 uM EC50 (doxorubicin alone) = 1.7 uMSK-OV-3 EC50 (prodrug alone) = 830 nM EC50 (prodrug + 25 nM FAP) = 140nM EC50 (prodrug + 100 uM 5057) = 56 uM EC50 (doxorubicin alone) = 440nM. Note: This assay involved a 72 hour, rather than the typical 48hour, incubation of prodrug with the cells prior to addition of theCellTiter-Blue reagent. 6. FAP digest of 5057DOX: [FAP] 4.00 × 10⁻⁹ MVmax 5.19 × 10⁻⁹ M/sec Km 1.11 × 10⁻⁵ M Kcat(Vmax/[E]) 1.30 sec⁻¹kcat/Km 1.17 × 10⁵ M⁻¹sec⁻¹ Note: This assay involved a 72 hour, ratherthan the typical 48 hour, incubation of prodrug with the cells prior toaddition of the CellTiter-Blue reagent. 7. FAP digest of 5057DOX and3099DOX: Dox standards; Daunorubicin internal standard [FAP] 1 nM Vmax9.80 × 10⁻¹⁰ M/sec Km 4.85 × 10⁻⁶ M Kcat 0.98 sec⁻¹ Kcat/Km 2.00 × 10⁵M⁻¹sec⁻¹ Note: This assay involved a 72 hour, rather than the typical 48hour, incubation of prodrug with the cells prior to addition of theCellTiter-Blue reagent. 8. CellTiter-Blue cell viability assay with HEK-mFAP cells: EC50 (prodrug alone) = 1.1 uM EC50 (prodrug + 100 uM 5057)= >100 uM EC50 (doxorubicin) = 400 nM Note: This assay involved a 72hour incubation of prodrug with the cells prior to addition of theCellTiter-Blue reagent. 5107DOX

CellTiter-Blue cell viability assay with HEK- mFAP cells: EC50 (prodrugalone) = 940 nM EC50 (prodrug + 100 uM 5057-2) = >100 uM EC50(doxorubicin) = 390 nM Note: This assay was performed with a 72 hourincubation of the compounds with the cells prior to addition of theCellTiter-Blue reagent 5061DOX

1. CellTiter-Blue cell viability assay with HEK- Mock and HEK-mFAPcells: HEK-Mock EC50 (prodrug alone) = >100 uM EC50 (prodrug + 100 uM5057) = >100 uM EC50 (prodrug + 25 nM FAP) = 280 nM EC50 (prodrug + 50nM PREP) = >100 uM EC50 (doxorubicin alone) = 92 nM HEK-mFAP EC50(prodrug alone) = >100 uM EC50 (prodrug + 100 uM 5057) = >100 uM EC50(doxorubicin alone) = 420 nM. Note: This assay involved a 72 hour,rather than the typical 48 hour, incubation of prodrug with the cellsprior to addition of the CellTitcr-Bluc reagent. 2. CellTiter-Blue cellviability assays: HEK-Mock EC50 (prodrug alone) = >100 uM EC50(prodrug + 25 nM FAP) = 250 nM HEK-mFAP EC50 (prodrug alone) = >100 uMEC50 (prodrug + 100 uM 5057) = >100 uM MCF-7 EC50 (prodrug alone) = >100uM EC50 (prodrug + 25 nM FAP) = >100 uM EC50 (prodrug + 100 uM 5057)= >100 uM EC50 (doxorubicin alone) = 54 uM OVCAR-3 EC50 (prodrug alone)= >100 uM EC50 (prodrug + 25 nM FAP) = 1.5 uM EC50 (prodrug + 100 uM5057) = >100 uM EC50 (doxorubicin alone) = 1.7 uM SK-OV-3 EC50 (prodrugalone) = >100 uM EC50 (prodrug + 25 nM FAP) = 900 nM EC50 (prodrug + 100uM 5057) = >100 uM EC50 (doxorubicin alone) = 440 nM. Note: All assaysinvolved a 72 hour, rather than the typical 48 hour, incubation ofprodrug with the cells prior to addition of the CellTiter-Bluc reagent.3. Inhibition of FAP activity on HEK-mFAP cells: IC50 = >l0 uM.

Example 16 Selectivity of FAP Over PREP

Recombinant enzyme (FAP or PREP) was combined, at reaction concentrationof 12 nM, 24 nM, or 48 nM, with 240 nM 5057DOX in FAP buffer (50 mMTris-HCl, pH 7.4, 140 mM NaCl) and incubated at 37° C. Reactions werestopped at 0, 10, 20, or 30 minutes by addition of equal volume of 10 MVal-boroPro (FAP inhibitor). Doxorubicin was measured by liquidchromatography/mass spectroscopy (LCMS). Representative results areshown in FIG. 12.

Example 17 Tissue Distribution of 5057DOX in HEK-FAP Mice

Tumor-bearing HEK-FAP mice were administered 2 mg/kg 5057DOX byintravenous injection. Mice were then euthanized 20 or 40 min followingadministration of 5057DOX and tissues were collected for analysis.Tumor, heart, lung, kidney, liver, muscle, spleen, stomach, smallintestine, large intestine, pancreas, brain, and bone marrow tissuesseparately were placed into lysis buffer, homogenized, vortexed,incubated on wet ice for 40 min, sonicated 3 times for 3 sec,centrifuged for 30 min at 4° C., and then lysates analyzed for prodrugand “warhead”. Representative results are shown in FIG. 17.

Example 18 Efficacy of 5057DOX in HEK-FAP Mice

HEK-FAP mice were administered 9 mg/kg 5057DOX or vehicle control byintravenous injection on day 33 after inoculation with tumor. Tumorvolumes were monitored daily. Representative results for mice with tumorvolumes less than 200 mm³ on day 33 (i.e., on the day of treatment with5057DOX) are shown in FIG. 18. Comparative data for 3099DOX is shown inFIG. 19.

INCORPORATION BY REFERENCE

All U.S. patents and U.S. and PCT published patent applicationsmentioned in the description above are incorporated by reference hereinin their entirety.

EQUIVALENTS

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

1. A prodrug represented by Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ represents(C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy, (C₁-C₁₀)alkyl-C(O)—(C₁-C₁₀)alkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₁₀)alkyl, aryl,aryl(C₁-C₁₀)alkyl, heteroaryl, or heteroaryl(C₁-C₁₀)alkyl, wherein anyR¹ is optionally substituted with one or more substituents independentlyselected from the group consisting of halo, hydroxy, carboxylate, cyano,amino, nitro, and thio (—SH); or —C(X)R¹ represents an N-terminallyblocked alpha amino acid residue, wherein X is O; R² represents H or a(C₁-C₆)alkyl; R³ represents H or a (C₁-C₆)alkyl; R⁴ is absent orrepresents a (C₁-C₆)alkyl, —OH, —NH₂, or halogen; X represents O or S; Lrepresents a bond, or —N(H)-L- represents a self-immolative linker; andCyt′ represents a radical of an anthracycline or derivative thereof. 2.The prodrug of claim 1, wherein L is a bond.
 3. The prodrug of claim 1,wherein the anthracycline or derivative thereof is doxorubicin.
 4. Theprodrug of claim 1, wherein L- is a self-immolative linker.
 5. Theprodrug of claim 4, wherein the anthracycline or derivative thereof isdoxorubicin.
 6. The prodrug of claim 1, wherein R² is H.
 7. The prodrugof claim 1, wherein R³ is a (C₁-C₆)alkyl.
 8. The prodrug of claim 1,wherein R³ is methyl, ethyl, propyl, or isopropyl.
 9. The prodrug ofclaim 1, wherein R³ is methyl.
 10. The prodrug of claim 1, wherein—C(X)R¹ is a moiety which, at physiological pH, reduces cellpermeability of the prodrug relative to the anthracycline or derivativethereof.
 11. The prodrug of claim 1, wherein —C(X)R¹ comprises one ormore functional groups that are ionized at physiological pH.
 12. Theprodrug of claim 1, wherein —C(X)R¹ is an acyl(C₁-C₁₀)alkyl substitutedwith one or more functional groups that are ionized at physiological pH.13. The prodrug of claim 12, wherein —C(X)R¹ is represented by theformula HO₂C—(C₁-C₁₀)alkyl-C(O)—.
 14. The prodrug of claim 12, wherein—C(X)R¹ is represented by the formula HO₂C—(CH₂)₂—C(O)—.
 15. The prodrugof claim 1, wherein —C(X)R¹ is selected from the group consisting offormyl, dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl, andmethoxysuccinyl.
 16. The prodrug of claim 1, wherein —C(X)R¹ is selectedfrom the group consisting of aryl(C₁-C₆)acyl and heteroaryl(C₁-C₆)acyl.17. The prodrug of claim 16, wherein —C(X)R¹ is an aryl(C₁-C₆)acyl. 18.The prodrug of claim 17, wherein the aryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with an aryl selected from the group consisting of benzyl,naphthalenyl, phenanthrenyl, phenolyl, and anilinyl.
 19. The prodrug ofclaim 18, wherein the aryl(C₁-C₆)acyl is a (C₁)acyl substituted with anaryl selected from the group consisting of benzyl, naphthalenyl,phenanthrenyl, phenolyl, and anilinyl.
 20. The prodrug of claim 16,wherein —C(X)R¹ is a heteroaryl(C₁-C₆)acyl.
 21. The prodrug of claim 20,wherein the heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acyl substituted with aheteroaryl selected from the group consisting of pyrryl, furyl,thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl,pyridinyl, pyrazinyl, pyridazinyl, and pyrimidinyl.
 22. The prodrug ofclaim 21, wherein the heteroaryl(C₁-C₆)acyl is a (C₁)acyl substitutedwith a heteroaryl selected from the group consisting of pyrryl, furyl,thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl,pyridinyl, pyrazinyl, pyridazinyl, and pyrimidinyl.
 23. The prodrug ofclaim 1, wherein —C(X)R¹ represents an N-terminally blocked alpha aminoacid residue, wherein X is O.
 24. The prodrug of claim 1, represented bya formula selected from the group consisting of:


25. The prodrug of claim 1, represented by the formula


26. A pharmaceutical composition, comprising a prodrug of claim 1, or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.
 27. A method of treating a disorder characterized byfibroblast activation protein (FAP) upregulation, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a prodrug of claim 1, or a pharmaceutically acceptable saltthereof. 28-31. (canceled)
 32. Use of a prodrug of claim 1, or apharmaceutically acceptable salt thereof, in the manufacture of amedicament for the treatment of a disorder characterized by fibroblastactivation protein (FAP) upregulation. 33-36. (canceled)